U.S. patent application number 10/225447 was filed with the patent office on 2004-02-26 for chemical reagents and methods for detection and quantification of proteins in complex mixtures.
Invention is credited to Aebersold, Rudolf H., Bottari, Patricia Q., Gelb, Michael H., Turecek, Frantisek.
Application Number | 20040038319 10/225447 |
Document ID | / |
Family ID | 31887009 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038319 |
Kind Code |
A1 |
Aebersold, Rudolf H. ; et
al. |
February 26, 2004 |
Chemical reagents and methods for detection and quantification of
proteins in complex mixtures
Abstract
The invention provides a reagent comprising an affinity tag, a
detectable moiety, a linker, an isotope tag and a reactive group.
The invention also provides methods of using a reagent of the
invention. The methods can be used to label a polypeptide in a
sample by contacting a sample with a reagent of the invention under
conditions allowing the reactive group to bind to one or more
polypeptides in the sample. The invention additionally provides
methods of isolating, identifying and quantifying a polypeptide in
a sample. The invention further provides methods of diagnosing a
disease using a reagent of the invention.
Inventors: |
Aebersold, Rudolf H.;
(Mercer Island, WA) ; Bottari, Patricia Q.;
(Seattle, WA) ; Gelb, Michael H.; (Seattle,
WA) ; Turecek, Frantisek; (Seattle, WA) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Family ID: |
31887009 |
Appl. No.: |
10/225447 |
Filed: |
August 20, 2002 |
Current U.S.
Class: |
435/7.5 ;
530/403 |
Current CPC
Class: |
G01N 33/585 20130101;
G01N 33/6809 20130101; G01N 33/6803 20130101 |
Class at
Publication: |
435/7.5 ;
530/403 |
International
Class: |
G01N 033/53; C07K
014/00 |
Goverment Interests
[0001] This invention was made with government support under grant
number GM60184 awarded by the National Institutes of Health. The
government has certain rights to the invention.
Claims
What is claimed is:
1. A reagent comprising an affinity tag, a detectable moiety, a
linker, an isotope tag and a reactive group.
2. The reagent of claim 1, wherein the detectable moiety is a
fluorophore or chromophore.
3. The reagent of claim 1, wherein the detectable moiety is
radioactive.
4. The reagent of claim 3, wherein the detectable moiety comprises
a radioactive atom selected from the group consisting of .sup.3H,
.sup.14C, .sup.32P, .sup.35S and .sup.125I.
5. The reagent of claim 1, wherein said linker is cleavable.
6. The reagent of claim 5, wherein said linker comprises a
photocleavable moiety, a chemically cleavable moiety, or an
enzymatically cleavable moiety.
7. The reagent of claim 1, wherein said affinity tag is selected
from the group consisting of biotin or a derivative thereof,
glutathione, maltose, poly(His), and an epitope tag.
8. The reagent of claim 1, wherein said reactive group is selected
from the group consisting of a sulfhydryl reactive group, an amine
reactive group, and a carboxyl reactive group.
9. The reagent of claim 1, said reagent having the general formula:
2wherein A is an affinity tag, D is a detectable moiety, L is a
linker, T is an isotope tag, and R is a reactive group.
10. The reagent of claim 9, wherein the detectable moiety is a
fluorophore or chromophore.
11. The reagent of claim 9, wherein the detectable moiety is
radioactive.
12. The reagent of claim 11, wherein the detectable moiety
comprises a radioactive atom selected from the group consisting of
.sup.3H, .sup.14C, .sup.32P, .sup.35S and .sup.125I.
13. The reagent of claim 9, wherein said linker is cleavable.
14. The reagent of claim 13, wherein said linker comprises a
photocleavable moiety, a chemically cleavable moiety, or an
enzymatically cleavable moiety.
15. The reagent of claim 9, wherein said affinity tag is selected
from the group consisting of biotin or a derivative thereof,
glutathione, maltose, poly(His), and an epitope tag.
16. The reagent of claim 9, wherein said reactive group is selected
from the group consisting of a sulfhydryl reactive group, an amine
reactive group, and a carboxyl reactive group.
17. A kit comprising the reagent of claim 1.
18. The kit of claim 17, wherein the detectable moiety of said
reagent is a fluorophore or chromophore.
19. The kit of claim 17, wherein the detectable moiety of said
reagent is radioactive.
20. The kit of claim 19, wherein the detectable moiety of said
reagent comprises a radioactive atom selected from the group
consisting of .sup.3H, .sup.14C, .sup.32P, .sup.35S and 125I.
21. The kit of claim 17, wherein said linker of said reagent is
cleavable.
22. The kit of claim 21, wherein said linker of said reagent
comprises a photocleavable moiety, a chemically cleavable moiety,
or an enzymatically cleavable moiety.
23. The kit of claim 17, wherein said affinity tag of said reagent
is selected from. the group consisting of biotin or a derivative
thereof, glutathione, maltose, poly(His), and an epitope tag.
24. The kit of claim 17, wherein said reactive group is selected
from the group consisting of a sulfhydryl reactive group, an amine
reactive group, and a carboxyl reactive group.
25. A method of labeling a polypeptide in a sample, comprising
contacting a sample with the reagent of claim 1 under conditions
allowing the reactive group to bind to one or more polypeptides in
said sample.
26. The method of claim 25, wherein the detectable moiety of said
reagent is a fluorophore or chromophore.
27. The method of claim 25, wherein the detectable moiety of said
reagent is radioactive.
28. The method of claim 27, wherein the detectable moiety of said
reagent comprises a radioactive atom selected from the group
consisting of .sup.3H, .sup.14C, .sup.32P, .sup.35S and
.sup.125I.
29. The method of claim 25, wherein said linker of said reagent is
cleavable.
30. The method of claim 29, wherein said linker of said reagent
comprises a photocleavable moiety, a chemically cleavable moiety,
or an enzymatically cleavable moiety.
31. The method of claim 25, wherein said affinity tag is selected
from the group consisting of biotin or a derivative thereof,
glutathione, maltose, poly(His), and an epitope tag.
32. The method of claim 25, wherein said reactive group is selected
from the group consisting of a sulfhydryl reactive group, an amine
reactive group, and a carboxyl reactive group.
33. A method of isolating a polypeptide in a sample, comprising the
steps of: (a) contacting a sample with the reagent of claim 1 under
conditions allowing the reactive group to bind to and react with
one or more polypeptides in said sample, thereby tagging one or
more polypeptides with said reagent; (b) resolving the polypeptides
in said sample; (c) visualizing the polypeptides tagged with said
reagent; (d) contacting said tagged polypeptides with a capture
moiety for said affinity tag; and (e) isolating said tagged
polypeptides.
34. The method of claim 33, wherein said linker of said reagent is
cleavable.
35. The method of claim 34, further comprising the step of cleaving
said linker.
36. The method of claim 35, wherein said linker of said reagent
comprises a photocleavable moiety, a chemically cleavable moiety,
or an enzymatically cleavable moiety.
37. The method of claim 33, wherein the detectable moiety of said
reagent is a fluorophore or chromophore.
38. The method of claim 33, wherein the detectable moiety of said
reagent is radioactive.
39. The method of claim 38, wherein the detectable moiety comprises
a radioactive atom selected from the group consisting of .sup.3H,
.sup.14C, .sup.32P, .sup.35S and 125I.
40. The method of claim 33, wherein said affinity tag of said
reagent is selected from the group consisting of biotin or a
derivative thereof, glutathione, maltose, poly(His), and an epitope
tag.
41. The method of claim 33, wherein said reactive group of said
reagent is selected from the group consisting of a sulfhydryl
reactive group, an amine reactive group, and a carboxyl reactive
group.
42. A method of identifying a polypeptide in a sample, comprising
the steps of: (a) contacting a sample with the reagent of claim 1
under conditions allowing the reactive group to bind to and react
with one or more polypeptides in said sample, thereby tagging one
or more polypeptides with said reagent; (b) resolving the
polypeptides in said sample; (c) visualizing the one or more
polypeptides tagged with said reagent; (d) contacting said one or
more tagged polypeptides with a capture moiety for said affinity
tag; (e) isolating said one or more tagged polypeptides; (f)
cleaving the linker of said reagent to release said one or more
tagged polypeptides; and (g) identifying a released tagged
polypeptide.
43. The method of claim 42, wherein said identifying step is
performed using mass spectrometry.
44. The method of claim 43, further comprising quantifying the
amount of said identified polypeptide.
45. The method of claim 42, wherein said linker of said reagent is
cleavable.
46. The method of claim 45, further comprising the step of cleaving
said linker.
47. The method of claim 46, wherein said linker of said reagent
comprises a photocleavable moiety, a chemically cleavable moiety,
or an enzymatically cleavable moiety.
48. The method of claim 46, wherein the detectable moiety is a
fluorophore or chromophore.
49. The method of claim 42, wherein the detectable moiety is
radioactive.
50. The method of claim 49, wherein the detectable moiety comprises
a radioactive atom selected from the group consisting of .sup.3H,
.sup.14C, .sup.32P, .sup.35S and 125I.
51. The method of claim 42, wherein said affinity tag of said
reagent is selected from the group consisting of biotin or a
derivative thereof, glutathione, maltose, poly(His), and an epitope
tag.
52. The method of claim 42, wherein said reactive group is selected
from the group consisting of a sulfhydryl reactive group, an amine
reactive group, and a carboxyl reactive group.
53. A method of identifying a polypeptide in a sample, comprising
the steps of: (a) contacting a sample with the reagent of claim 1
under conditions allowing the reactive group to bind to and react
with one or more polypeptides in said sample, thereby labeling one
or more polypeptides with said reagent; (b) cleaving the
polypeptides in said sample to generate peptide fragments; (c)
adding one or more peptide standards to said sample, wherein said
peptide standards correspond to peptides generated from cleaving
sample polypeptides as in step (b) and are labeled with an
isotopically distinct version of said isotope tag; (d) resolving
the labeled peptides in said sample; (e) visualizing the labeled
peptides; (f) contacting said labeled peptides with a capture
moiety for said affinity tag; (g) isolating said labeled peptides;
(h) cleaving the linker of said reagent to release said isolated
peptides; and (i) identifying a released peptide.
54. The method of claim 53, wherein said identifying step is
performed using mass spectrometry.
55. The method of claim 53, further comprising quantifying the
amount of said identified peptide.
56. The method of claim 53, wherein said linker of said reagent is
cleavable.
57. The method of claim 56, further comprising the step of cleaving
said linker.
58. The method of claim 57, wherein said linker of said reagent
comprises a photocleavable moiety, a chemically cleavable moiety,
or an enzymatically cleavable moiety.
59. The method of claim 53, wherein the detectable moiety of said
reagent is a fluorophore or chromophore.
60. The method of claim 53, wherein the detectable moiety of said
reagent is radioactive.
61. The method of claim 60, wherein the detectable moiety comprises
a radioactive atom selected from the group consisting of .sup.3H,
.sup.14C, .sup.32P, .sup.35S and 125I.
62. The method of claim 53, wherein said affinity tag of said
reagent is selected from the group consisting of biotin or a
derivative thereof, glutathione, maltose, poly(His), and an epitope
tag.
63. The method of claim 53, wherein said reactive group of said
reagent is selected from the group consisting of a sulfhydryl
reactive group, an amine reactive group, and a carboxyl reactive
group.
64. The method of claim 53, wherein the polypeptides in said sample
are cleaved with trypsin.
65. A method of diagnosing a disease, comprising the steps of: (a)
contacting a sample from a patient with the reagent of claim 1
under conditions allowing the reactive group to bind to and react
with one or more polypeptides in said sample, thereby labeling one
or more polypeptides with said reagent; (b) cleaving the
polypeptides in said sample to generate peptide fragments; (c)
adding one or more peptide standards to said sample, wherein said
peptide standards correspond to peptides generated from cleaving
sample polypeptides as in step (b), are labeled with an
isotopically distinct version of said isotope tag, and are derived
from a polypeptide exhibiting aberrant expression in said disease;
(d) resolving the labeled peptides in said sample; (e) visualizing
the labeled peptides; (f) contacting the labeled peptides with a
capture moiety for said affinity tag; (g) isolating said labeled
peptides; (h) cleaving the linker of said reagent to release said
isolated peptides; (j) quantifying said released peptides; and (k)
comparing the amount of sample peptide to standard peptide in said
released peptides, wherein the amount of sample peptide is
correlated with the diagnosis of said disease.
66. The method of claim 65, wherein said disease is muscular
dystrophy.
67. The method of claim 66, wherein said disease is Duchenne
muscular dystrophy or Becker muscular dystrophy.
68. The method of claim 65, wherein said disease is prostate
cancer.
69. The method of claim 65, wherein said identifying step is
performed using mass spectrometry.
70. The method of claim 65, wherein said linker is cleavable.
71. The method of claim 70, further comprising the step of cleaving
said linker.
72. The method of claim 65, wherein said linker of said reagent
comprises a photocleavable moiety, a chemically cleavable moiety,
or an enzymatically cleavable moiety.
73. The method of claim 65, wherein the detectable moiety of said
reagent is a fluorophore or chromophore.
74. The method of claim 65, wherein the detectable moiety of said
reagent is radioactive.
75. The method of claim 74, wherein the detectable moiety comprises
a radioactive atom selected from the group consisting of .sup.3H,
.sup.14C, .sup.32P, .sup.35S and 125I.
76. The method of claim 65, wherein said affinity tag of said
reagent is selected from the group consisting of biotin or a
derivative thereof, glutathione, maltose, poly(His), and an epitope
tag.
77. The method of claim 65, wherein said reactive group of said
reagent is selected from the group consisting of a sulfhydryl
reactive group, an amine reactive group, and a carboxyl reactive
group.
78. The method of claim 65, wherein the polypeptides in said sample
are cleaved with trypsin.
Description
BACKGROUND OF THE INVENTION
[0002] The invention is related to the field of analytical
biochemistry, and more specifically to the field of quantitative
proteomics.
[0003] Large scale sequencing of genomic DNA, cDNA and EST's
(expressed sequence tags) has identified large numbers of genes.
For some selected species, the complete genome sequences have been
determined and, for others, EST sequence databases are assumed to
contain sequence tags for the majority of genes for that species.
For the species having complete genome sequences or substantial EST
sequences available, essentially all the genes have been
determined. Other genomic technologies including serial analysis of
expressed genes (SAGE) and differential cDNA or oligonucleotide
array analysis are capable of determining quantitative expression
profiles of the expressed genes at the mRNA level in specific cells
or tissues. Furthermore, comparative genomic analyses have
identified genes that, if defective or aberrantly expressed, are
the cause of genetic or somatic diseases.
[0004] From genomic analysis or the analysis of the expressed mRNA
transcripts neither the quantity nor the structure, activity and
state of modification of the translated protein products can be
predicted. Recently developed mass spectrometric techniques allow
the rapid identification of expressed proteins by the correlation
of mass spectral data that are idiotypic for the sequence of a
specific protein with the sequences contained in sequence
databases.
[0005] A number of approaches have been used to address the needs
of proteomics analysis, including the use of reagents that allow
relative quantitation of proteins in a sample. However, none of the
previously described methods can be used to determine the absolute
quantity of the proteins in a sample. Furthermore, these methods do
not allow the analysis to be focused on selected proteins such as
those that are involved in specific diseases or those that are
predicted or expected to be present based on circumstantial data or
considerations.
[0006] Thus, there exists a need for reagents suitable for absolute
quantitative analysis of protein samples as well as reagents
suitable for analysis of selected proteins, which can be used as
diagnostic or prognostic markers. The present invention satisfies
these needs, and provides related advantages as well.
SUMMARY OF THE INVENTION
[0007] The invention provides a reagent comprising an affinity tag,
a detectable moiety, a linker, an isotope tag and a reactive group,
referred to herein as a VICAT reagent. The invention also provides
methods of using a reagent of the invention. The methods can be
used to label a polypeptide in a sample by contacting a sample with
a reagent of the invention under conditions allowing the reactive
group to bind to one or more polypeptides in the sample. The
invention additionally provides methods of isolating, identifying
and quantifying a polypeptide in a sample. The invention further
provides methods of diagnosing a disease using a reagent of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1A shows the structure and synthetic scheme of a VICAT
reagent having 7- nitrobenz-2-oxa-1,3-diazole (NBD) as the
detectable moiety. FIG. 1B shows a detailed reaction scheme for the
synthesis-of a VICAT reagent having NBD as the detectable moiety.
The structures of intermediate compounds 1-7 and final product,
compound 8 (VICAT having NBD as detectable moiety), are shown.
[0009] FIG. 2 shows the structure and synthetic scheme of a VICAT
reagent having tetramethyl-rhodamine as the detectable moiety.
[0010] FIG. 3 shows photocleavage of a VICAT reagent-peptide
conjugate. A portion of a VICAT reagent depicted in FIG. 2 is shown
conjugated to a peptide. The VICAT-peptide conjugate undergoes
photocleavage to generate a VICAT fragment and a peptide
intermediate. The peptide intermediate spontaneously gives of
CO.sub.2 gas, yielding a NAMETAG-peptide conjugate as the final
product. The NAMETAG portion can contain heavy isotopic
substitution, as exemplified in FIG. 2.
[0011] FIG. 4A shows the structure of a VICAT reagent having a
radioactive acetyl group as the detectable moiety
(.sup.14C-Acetyl-VICAT). FIG. 4B shows a reaction scheme for the
synthesis of .sup.14C-Acetyl-VICAT.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The invention provides reagents and methods for the
detection of specific proteins in a complex sample. The methods can
be used to quantify the amount of protein in the sample. The method
is based on the concurrent introduction of a detectable moiety, for
example, a fluorescent reagent or a radiolabel, a stable isotope
tag, and an affinity tag at specific sites in a protein. The
detectable moiety is used for the visualization of tagged peptides
after proteolysis of the tagged protein sample and peptide
separation. The stable isotope tag is used for accurate
quantification of the abundance of the protein in the sample using
a mass spectrometer. The affinity tag is required for the isolation
of the tagged peptides. The reagent having these properties is
termed VICAT, for Visible Isotope-coded Affinity Tag. To avoid
complications in analyzing tandem mass spectra of tagged peptides
caused by the relatively large VICAT group, the reagents can
contain a cleavable linker for the removal of a major portion of
the VICAT group prior to mass spectrometric analysis. The reagents
and methods of the invention allow the rapid, sensitive and
quantitative detection, as well as accurate absolute
quantification, of selected proteins contained in complex
samples.
[0013] A VICAT reagent of the invention has the following general
structure: i) a functional group that specifically reacts with a
chemical group in the target molecule, which is typically a
protein; ii) a stable isotope tag that remains covalently attached
to the specific target molecule and peptides derived from the
target molecule; iii) a cleavable linker peripheral to the isotope
tag; iv) a detectable group; and v) an affinity group.
[0014] The methods of the invention advantageously allow the
detection of one or several selected proteins in complex samples
and determination of their absolute quantity. In general, the
method is based on the generation of labeled peptide standards for
each target protein, where the peptide standards are chemically
identical to, but isotopically distinguished from, the VICAT
labeled peptides generated by enzymatic digestion of a protein
sample mixture containing the target protein. The standard peptides
are generated by chemical synthesis of the target peptide followed
by labeling of the peptide with one isotopic form of the VICAT
reagent. The standard peptides conjugated with a VICAT reagent are
purified and can be quantitatively calibrated for use as a known
standard.
[0015] Defined amounts of the labeled peptide standards are added
to the protease digest of the sample, and the peptide mixture is
separated by isoelectric focusing (IEF) in a polyacrylamide gel or
in solution, for example, in a capillary. Other types of peptide
separation can also be used, for example, reverse phase high
pressure liquid chromatography (HPLC) or other well known methods
of peptide separation, as disclosed herein. The location of the
tagged peptides in the gel or capillary or other form of peptide
separation is determined by fluorescence or visual imaging of a
similarly prepared isoelectric focusing strip or capillary that
contains only the VICAT-tagged peptide standards, or by
autoradioagraphy or counting of radioactive fractions of the gel or
capillary if the detectable moiety of the VICAT reagent is a
radiolabel. The region of the sample-containing strip where the
tagged peptides are located is extracted, and the tagged peptides
are immobilized to affinity isolate the tagged peptides. The
peptides are affinity isolated by contacting the tagged peptides
with a solid support containing the cognate binding molecule of the
affinity group of the VICAT reagent, for example, using avidin or
streptavidin to bind to biotin on the VICAT reagent. Immobilized
peptides are recovered either by adding a competitive ligand for
the affinity group of the VICAT reagent, by changing the solution
conditions such that the VICAT-peptide conjugate is released from
the solid support, for example, by changing pH, salt, chaotropic
agents, and the like, or by cleavage of a cleavable linker, leaving
the isotope tag covalently attached to the peptide. The recovered
peptides can be analyzed by mass spectrometry and, if required, by
tandem mass spectrometry. Accurate protein quantification is
achieved by calculating the ratio of signal intensities of the
standard peptide and the differentially labeled peptide of
identical sequence generated by enzymatic digestion of the protein
sample.
[0016] Although described above with the affinity isolation step
performed after a previous purification step, it is understood that
the affinity isolation step of VICAT-labeled polypeptides can be
performed at any convenient step of the methods of the invention.
For example, the affinity isolation step can be performed prior to
resolution on an IEF gel or chromatographic separation.
Furthermore, the affinity isolation step can be performed prior to
digestion with a protease, if desired. One skilled in the art can
readily determine a suitable order for carrying out steps of the
methods of the invention so long as VICAT-labeled peptides are
isolated.
[0017] The method of the invention involves the separation of the
peptide mixtures generated by the digestion of the labeled protein
samples. Among the multitude of mature peptide separation methods
available, a particularly useful method is isoelectric focusing in
polyacrylamide gels with immobilized pH gradients. This method is
particularly useful for the following reasons: i) it is robust,
relatively inexpensive, highly reproducible and easily multiplexed.
IEF strips covering different pH ranges are commercially available;
ii) the method has immense separation power and the pH gradient and
therefore the range of maximal separation can be tuned; iii) the pI
of peptides can be reasonably accurately calculated from the amino
acid sequence. The position of a specific target peptide in the gel
can therefore be predicted; and iv) peptide recovery is simple and
efficient.
[0018] The methods of the invention can be advantageously used for
the detection and accurate absolute quantification for one or more
particular proteins in a complex protein sample. The methods for
detecting and quantitating proteins or other molecules in a sample
can be used for a variety of purposes. For example, the methods of
the invention can be used for the detection and quantification of
proteins coded for by genetic disease genes in cell lysates or
other protein extracts prepared from biopsies or body fluids for
the purpose of diagnosis or prognosis. The methods of the invention
are also useful for the detection and quantification of
specifically modified forms of a protein. In addition, the methods
of the invention can be used for the detection and quantification
of sequence variants of a protein, including polymorphisms, splice
isoforms and sequence variants created by post-translational
processing. Furthermore, the methods of the invention can be used
for the detection of a single or multiple marker proteins in a
biological sample, including those that are diagnostic or
prognostic for a specific disease. The methods of the invention are
advantageous in allowing quantification of the absolute amounts of
multiple proteins in a complex sample without the need for specific
reagents such as antibodies and can be carried out within a
simplified operation.
[0019] Moreover, because the methods can be used quantitatively,
the methods of the invention can be used for the detection of
changes in abundance of specific proteins in complex mixtures. For
example, the detection of proteins that are aberrantly expressed in
disease states, genetic or otherwise, that is, the detection of
marker proteins, is common in clinical testing and diagnosis.
Commonly, such tests are based on some form of immunodetection, for
example, western blotting, ELISA, of the target protein(s), which
requires one or several antibodies specific for the target protein,
and typically one protein is measured per assay. In contrast, the
methods of the invention are advantageous in that they allow the
detection of changes in the abundance of a selected single protein
or constellations of multiple proteins in complex samples.
[0020] The methods of the invention incorporate a modification of
the previously described ICAT-based proteomics technology (Gygi et
al., Nat. Biotechnol. 17:994-999 (1999); WO 00/11208, each of which
is incorporated herein by reference). The use of an ICAT-type
reagent is advantageous for detecting one or several proteins of
known identity in a complex protein mixture and can be greatly
simplified by the ability to readily prepare an internal standard
that is chemically identical, but isotopically-distinguished, to
the tagged peptides derived from the proteins of interest. The
methods of the invention using a VICAT-type reagent advantageously
allow the ready detection of the protein of interest using a
detectable group such as a fluorophore or radiolabel. Thus, an
ICAT-type reagent can be modified to contain a fluorophore, a
visible dye, or radiolabel so that the internal standard can be
readily detected and quantitatively calibrated either
fluorimetrically or simply by eye in the case of colored
fluorophores or dyes, or by autoradiography, liquid scintiallation
counting or Cerenkov counting in the case of radiolabels. Since the
target proteins are generally characterized using high-resolution
chromatographic separation of peptides prior to their detection by
ESI-MS, the reagents of the invention allow the chromatographic
position of the tagged peptides of interests to be readily
visualized.
[0021] Thus, Visible ICAT reagents (VICAT reagents) are prepared
having the properties of an ICAT reagent (Gygi et al., supra, 1999;
WO 00/11208). The VICAT-type reagent also contains a detectable
moiety such as a fluorophore or radiolabel. A VICAT-type reagent
also can include a cleavable linker. The inclusion of a cleavable
linker allows release of a significant portion of the VICAT reagent
after chromatographic separation and visualization of the
VICAT-tagged protein of interest. After cleavage, a small,
isotopically substituted fragment of the VICAT reagent remains
covalently attached to the protein or peptide of interest. This
allows the peptide from which the bulk of the VICAT reagent has
been removed to be analyzed by tandem ESI-MS with the ion
fragmentation directed in the peptide rather than in the tag.
Peptide-directed fragmentation allows for peptide sequencing and
thus peptide identification.
[0022] The reagents and methods of the invention have widespread
application in quantitative biochemistry and proteomics. Among the
advantages of the methods of the invention are the potential
ability to detect and quantify multiple peptides in the same
analysis (multiplexing), very precise quantification, and the lack
of need for peptide-specific antibodies or other reagents.
[0023] The invention provides a VICAT reagent comprising an
affinity tag, a detectable moiety, such as a fluorescent or
non-fluorescent dye or radiolabel, a linker, which can be
cleavable, a stable isotope tag, and a reactive group. The VICAT
reagent is used to tag a molecule such as a polypeptide to modify
the molecule with specific functionalities. In particular, the
reactive group is a chemical moiety having reactivity for a
chemical moiety on the target molecule. Thus, the reactive group
couples the VICAT reagent to a target molecule. Once tagged with a
VICAT reagent, the target molecule is attached to an affinity tag,
a detectable moiety, a linker and an isotope tag. The affinity tag
can be used to specifically isolate tagged molecules. The
detectable group is used to visualize or locate tagged molecules.
The linker serves to bridge the affinity tag and detectable group
to the isotope tag. The linker can include a cleavable moiety so
that the tagged molecule can be released from the affinity tag and
detectable moiety. For example, if the affinity tag is used to
affinity isolate tagged molecules on a solid support, the linker
can be cleaved to release the affinity isolated tagged
molecules.
[0024] The isotope tag is a chemical moiety that can be used to
incorporate heavy isotopes, allowing the generation of multiple
forms of a chemically identical but isotopically distinct moiety.
Differential isotope tags are particularly useful for quantitative
analysis since a sample molecule can be compared directly to a
standard molecule, with the only difference being the differential
isotope tag, thus allowing direct comparison between the sample and
standard molecule. Due to the relative position of the
functionalities of a VICAT reagent of the invention, the VICAT
reagent can be used to affinity isolate peptides and release the
peptides from the affinity support while concurrently transferring
an isotope tag to the released peptides. Incorporation of the
isotope tag allows comparison to standard peptides labeled with a
differentially labeled isotope tag for convenient qualitative
and/or quantitative analysis.
[0025] An exemplary VICAT reagent of the invention can have the
general formula: 1
[0026] where A is an affinity tag, D is a detectable moiety, such
as a fluorescent dye or radiolabel, L is a linker, which can be
cleavable, T is an isotope tag, and R is a reactive group having a
chemical functionality suitable for covalently coupling to a target
molecule such as a protein. The formula above is representative of
the VICAT reagents shown in FIGS. 1, 2 and 4. It is understood that
any arrangement of the functionalities of the VICAT reagent can be
employed so long as each of the moieties can function as disclosed
herein and needed for a particular application. Generally, the
affinity tag and detectable moiety are positioned so that the
linker separates them from the isotope tag and reactive group,
allowing cleavage of the affinity tag and detectable moiety from a
tagged molecule. Thus, in addition to the formula shown above, a
reagent of the invention can have the formula A-D-L-T-R or
D-A-L-T-R, or any arrangement that allows the affinity tag,
detectable moiety, linker, isotope tag, and reactive group to
function as desired.
[0027] The structures and synthetic schemes of exemplary VICAT
reagents are shown in FIGS. 1, 2 and 4. The reactive functional
group depicted in FIGS. 1, 2 and 4 is an iodoacetyl group, which
specifically reacts with SH groups on reduced proteins. The
particular isotope tag group depicted is an N-aminoethylacetamido
group, also known as a NAMETAG group. The linker shown is a
cleavable linker containing the photocleavable
2-(2-nitrophenyl)-ethan-2-ol group. The dye group shown in FIG. 1
is 7- nitrobenz-2-oxa-1,3-diazole (NBD). The dye group shown in
FIG. 2 is 5-carboxytetramethylrhodamine. The affinity group shown
is a biotin. FIG. 3 shows how the NAMETAG-peptide is formed after
photocleavage of a VICAT-peptide conjugate.
[0028] The VICAT reagents described above and depicted in FIGS. 1,
2 and 4 are merely exemplary of the reagent of the invention having
an affinity tag, a detectable moiety, a linker, and isotope tag,
and a reactive group, which can have the general formulas described
above. One skilled in the art will readily recognize that many
structural variations of the structures shown in FIGS. 1, 2 and 4
would be suitable for a reagent of the invention and would provide
the above-described characteristics.
[0029] In particular, an affinity tag of the VICAT reagent is used
so that, when the VICAT reagent is attached to a molecule, the
affinity tag can be used to isolate the molecule from a complex
mixture. As used herein, an "affinity tag" refers to a chemical
moiety having binding activity for a capture moiety. One skilled in
the art can readily determine a suitable affinity tag for use in a
VICAT reagent of the invention. The general features of an affinity
tag include being stable, relatively small, having high affinity
and specific binding activity to a cognate binding partner, being
unlikely to undergo non-specific interactions and adsorption to
vessels, and being soluble in aqueous solution. Accordingly,
suitable criteria for selection of an affinity tag include but are
not limited to a desired size, solubility, affinity and/or
specificity for a particular capture moiety, lack of non-specific
binding, the nature of the molecule to be captured, such as whether
any of the sample molecules would be able to bind to the capture
moiety, and the like. Any of these or other desirable criteria can
be considered by one skilled in the art for affinity tag to be
incorporated into a VICAT reagent of the invention. An exemplary
affinity tag is biotin, and the corresponding capture moiety is
avidin or streptavidin. Biotin is particularly useful as an
affinity tag because it forms a high affinity interaction with
avidin or streptavidin and can therefore readily be used to
affinity isolate molecules that have reacted with the VICAT
reagent.
[0030] In addition to biotin, other affinity tags can be used (see
WO 00/11208). For example, modified biotin-based reagents can be
used, including d-iminobiotin, which bind to avidin/streptavidin.
Another useful biotin-based reagent is N-biotinyl-sarcosine, which
binds tightly to avidin/streptavidin but can be released more
easily than biotin (see Gerber et al., Bioconj. Chem. 12:603-615
(2001)). Maltose can also be used as an affinity tag since it binds
to maltose binding protein. Similarly, other sugars and sugar
binding molecules such as lectins are suitable as an affinity
tag/capture moiety pair. Another suitable affinity tag/capture
moiety pair includes glutathione, which binds to
glutathione-S-transferase. In addition, a poly-His tag, generally
containing about 5 to about 10 histidines, can be used to affinity
purify a molecule using metal chelate chromatography.
[0031] Other suitable affinity tags include an epitope for which a
specific antibody is available. An epitope can be, for example, a
short peptide of about 3-5 amino acids or more, a carbohydrate, a
small organic molecule, and the like. Epitope tags have been used
to affinity purify recombinant proteins and are commercially
available. For example, antibodies to epitope tags, including myc,
FLAG, hemaglutinin (HA), green fluorescent protein (GFP), polyHis,
and the like, are commercially available (see, for example, Sigma,
St. Louis Mo.; PerkinElmer Life Sciences, Boston Mass.). In
addition, a hapten, such as a dinitrophenyl group, can be used with
an antibody specific for the hapten. Methods for making antibodies
against an epitope tag are well known to those skilled in the art
(Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press (1988)). One skilled in the art can readily
determine suitable affinity tags for isolation of molecules to
which the VICAT reagent has been attached.
[0032] Yet another suitable affinity tag is a 1,2-diol, such as
1,2-dihyroxyalkanes, including those of cyclic alkanes, for
example, 1,2-dihydroxycyclohexane, which bind to an alkyl or aryl
boronic acid or boronic acid esters , such as phenyl-B(OH).sub.2 or
hexyl-B(OEthyl).sub.2, which can be attached via the alkyl or aryl
group to a solid support material, such as agarose. A
nitrilotriacetic acid group can also be used as an affinity tag (WO
00/11208).
[0033] The above-described affinity tags can interact
non-covalently with the corresponding capture moiety.
Alternatively, an affinity tag can interact covalently with a
capture moiety. In such a case, either the affinity tag or the
capture moiety, in addition to containing chemical characteristics
providing a specific interaction, contains a reactive group capable
of reacting covalently to join the affinity tag and capture moiety.
Such a covalent interaction would allow stringent wash conditions,
and the presence of a cleavable linker in the VICAT reagent would
allow release and tagging of the VICAT-labeled molecule(s), as
disclosed herein.
[0034] Furthermore, it is understood that the affinity tag of the
VICAT reagent need not be distinct from other moieties. If a
capture moiety is available for another moiety of a VICAT reagent,
then one moiety of the VICAT reagent can serve two functions. For
example, antibodies are available against the detectable moiety
fluorescein, and fluorescein can thus function as both an affinity
tag and detectable moiety. Similarly, other moieties can have
multiple functions in a VICAT reagent so long as each of the
functions can work independently.
[0035] The VICAT-type reagents of the invention also include a
detectable moiety. As used herein, a "detectable moiety" refers to
a moiety that imparts a physicochemical property such that the
moiety can be detected. Such a detectable moiety allows a
VICAT-tagged molecule to be visualized. The detectable moiety is
generally a moiety that allows convenient detection of the tagged
molecule, for example, a fluorescent, a colored, or a calorimetric
moiety. Similar to the desired characteristics of an affinity tag,
the detectable moiety is generally stable, relatively small, is
unlikely to undergo non-specific interactions and adsorption to
vessels, and is soluble in aqueous solution. In the case of a
fluorescent moiety, it is particularly useful for the moiety to
have high quantum yield, a high absorption coefficient, and a
quantum yield that is not solvent dependent. Accordingly, suitable
criteria for selection of a detectable moiety include but are not
limited to a desired size, solubility, lack of non-specific
binding, desired method of detection including speed and
convenience of detection, and the like. In the case of fluorescent
detectable moieties, the criteria can include quantum yield and
properties related to solvent, absorption coefficient, excitation
and emission wavelengths, fluorescence lifetime, and the like. Any
of these or other desirable criteria can be considered by one
skilled in the art for affinity tag to be incorporated into a VICAT
reagent of the invention.
[0036] Thus, the detectable group can be a fluorophore, such as
rhodamine-type fluorophore depicted in FIG. 2. Other suitable
fluorescent moieties are well known to those skilled in the art
(see Hermanson, Bioconjugate Techniques, pp. 297-364, Academic
Press, San Diego (1996); Molecular Probes, Eugene Oreg.). Rhodamine
derivatives include, for example, tetramethylrhodamine, rhodamine
B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101),
rhodamine 110, and derivatives thereof such as
tetramethylrhodamine-5- (or 6), lissamine rhodamine B, and the
like. Other suitable fluors for a VICAT reagent include 7-
nitrobenz-2-oxa-1,3-diazole (NBD)(see FIG. 1).
[0037] Additional exemplary fluorophores include, for example,
fluorescein and derivatives thereof. Other flurophores include
napthalenes such as dansyl (5-dimethylaminonapthalene-1-sulfonyl).
Additional fluorophores include coumarin derivatives such as
7-amino-4-methylcoumarin-3-acetic acid (AMCA),
7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoum- arin
(DCIA), Alexa fluor dyes (Molecular Probes), and the like.
[0038] Other fluorophores include
4,4-difluoro-4-bora-3a,4a-diaza-s-indace- ne (BODIPY.TM.) and
derivatives thereof (Molecular Probes; Eugene Oreg.). Further
fluorophores include pyrenes and sulfonated pyrenes such as Cascade
Blue.TM. and derivatives thereof, including
8-methoxypyrene-1,3,6-trisulfonic acid, and the like. Additional
fluorophores include pyridyloxazole derivatives and dapoxyl
derivatives (Molecular Probes). Additional fluorophores include
Lucifer Yellow (3,6-disulfonate-4-amino-naphthalimide) and
derivatives thereof. CyDye.TM. fluorescent dyes (Amersham Pharmacia
Biotech; Piscataway N.J.) can also be used.
[0039] In general, the detectable moiety is a relatively small
organic molecule such as the fluorophores described above. However,
it is understood that fluorescent proteins can also be used so long
as the size of the fluorescent protein does not interfere with the
other functions of the VICAT reagent or the needs of the particular
experiment. Thus, other fluorescent moieties include
phycobiliproteins and derivatives such as phycoerythrin and
phycocyanin from various species, as well as green fluorescent
protein and derivatives thereof or other fluorescent proteins.
[0040] The detectable moiety can also be a chromophore. Exemplary
chromophores include, for example, phenolphthalein, malachite
green, nitroaromatics such as nitrophenyl, diazo dyes, dabsyl
(4-dimethylaminoazobenzene-4'-sulfonyl), and the like (see
Hermanson, supra, 1996).
[0041] Although the detectable moiety is described above as a group
that allows ready visualization of the VICAT-tagged molecule, it is
understood that any moiety that imparts a detectable property can
be used as a detectable moiety. Thus, a detectable moiety can be a
radioactive moiety, which can be visualized by autoradiography,
fluorography or phosphorimaging, or can be detected by
scintillation or Cerenkov counting of chromatographic fractions,
polyacrylamide gel or gel slices, or extracts of regions of a
polyacrylamide gel. Suitable radioactive isotopes for use in a
detectable moiety include, for example, .sup.3H, .sup.14C,
.sup.32P, .sup.35S, 125I, and the like. Thus, a radioactive
detectable moiety can include a chemical group that contains
hydrogen, carbon, phosphorus, sulfur, iodine, and the like
incorporated as a radioactive isotope. In addition, the detectable
moiety can be chemiluminescent, can contain spin labels, can be
detectable with ultraviolet light, or can be other detectable
groups.
[0042] It is understood that, when a detectable moiety is a small
moiety such as a radioactive atom, the detectable moiety can be
incorporated into one of the other functional groups of a VICAT
reagent. For example, a radioactive moiety can be attached to a
linker as a separate chemical moiety or can be incorporated into
the linker by using a radioactive atom in the linker. Similarly,
one or more radioactive atoms can be incorporated into the affinity
tag, isotope tag or reactive group. In such a case, the detectable
moiety can be positioned so that it is removed upon cleavage of the
linker. However, since the detectable moiety is relatively small,
it can be positioned so that the detectable moiety is retained on
the labeled polypeptide after cleavage of the linker and release
from an affinity capture reagent. An exemplary radioactive VICAT
reagent is depicted in FIG. 4.
[0043] The "linker" of a VICAT reagent is used to bridge the
affinity tag and detectable moiety to the isotope tag and reactive
group. The linker is generally a cleavable group. A cleavable group
is particularly useful when mass spectrometry (MS) is being used as
an analytical method. Because the affinity tag and detectable tag
can be large moieties, it is useful to have the option of cleaving
off these large groups from the molecules to be analyzed by MS.
However, it is understood that cleavage of a cleavable linker or
the use of a cleavable linker is not required so long as the groups
of the VICAT reagent retained on the tagged molecule are compatible
with the analytical method being used. Any of a number of chemical
groups can be used to bridge the affinity tag and detectable moiety
with the isotope tag and reactive group. Suitable criteria for
selection of a linker include but are not limited to a desired
size, solubility, ability to cleave or not cleave the linker, the
type of cleavage method to be used, and the like. Any of these or
other desirable criteria can be considered by one skilled in the
art for affinity tag to be incorporated into a VICAT reagent of the
invention.
[0044] A cleavable linker can be a photocleavable or chemically
cleavable moiety. The photocleavable linker
2-(2-nitrophenyl)-ethan-2-ol is depicted in FIGS. 1, 2 and 4. Other
suitable photocleavable linkers include, for example, linkers
containing o-nitrobenzyl, desyl, trans-o-cinnamoyl, m-nitrophenyl,
benzylsulfonyl groups (see, for example, Dorman and Prestwich,
Trends Biotech. 18:64-77 (2000); Greene and Wuts, Protective Groups
in Organic Synthesis, 2nd ed., John Wiley & Sons, New York
(1991); U.S. Pat. Nos. 5,143,854; 5,986,076; 5,917,016; 5,489,678;
5,405,783). One skilled in the art will readily recognize that
these exemplary photocleavable groups as well as others can be used
in a VICAT reagent.
[0045] In addition to a photocleavable linker, a chemically
cleavable linker can also be used. Exemplary cleavable linkers can
contain a disulfide, which can be cleaved with reducing agents; a
diol, which can be cleaved with periodate; a diazo bond, which can
be cleaved with dithionate; an ester, which can be cleaved with
hydroxylamine; and a sulfone, which can be cleaved with base (see
Hermanson, Bioconjugate Techniques, Academic Press, San Diego
(1996); Pierce Chemical Co., Rockford Ill.).
[0046] Furthermore, a linker cleavable by an enzyme can be used.
For example, a protease can be used to cleave a cleavable linker
having a suitable recognition sequence for the protease.
Particularly useful proteases are endopeptidases such as factor Xa,
tobacco etch virus (TEV) protease, trypsin, chymotrypsin,
Staphylococcus aureus protease, submaxillaris protease, and the
like. The protease can be selected based on the incorporation of a
particular cleavable recognition sequence into the linker. Other
considerations for selecting a protease include the presence or
absence of a recognition sequence in the molecule being captured
and released.
[0047] For example, a rare cleaving protease such as TEV protease
or factor Xa can be used to cleave a functional group containing
the corresponding protease recognition sequence, resulting in
release of the captured molecule. Such rare cleaving proteases are
particularly useful for releasing an intact polypeptide molecule
since the recognition sequence for these proteases would not occur
in the vast majority of polypeptides. Alternatively, a polypeptide
sample can be treated with a specific protease, and the digested
peptides isolated by the methods disclosed herein. In such a case,
the captured peptides would not contain a recognition sequence for
the protease used for cleavage since the polypeptide has already
been digested. In addition, if desired, an intact polypeptide can
be captured and digested with a protease after binding to the solid
support, resulting in the incorporation and release of a label on
the peptide fragment of the polypeptide that was captured on the
solid support. Thus, protease digestion can be used before or after
capture of a sample molecule, as desired.
[0048] In addition to proteases, a cleavable functional group can
be a recognition sequence for an endonuclease such as a restriction
enzyme. Thus, an appropriate recognition sequence for a restriction
enzyme can be incorporated as a cleavable functional group and
cleaved with the respective restriction enzyme. It is understood
that such a nucleotide functional group can be useful for capturing
and releasing a nucleic acid or a polypeptide, or any other type of
molecule, as desired. Similarly, a protease recognition sequence
can be useful for capturing and releasing a polypeptide, nucleic
acid or any other type of molecule, as desired.
[0049] A VICAT reagent of the invention also contains a stable
isotope tag. As used herein an "stable isotope tag" refers to a
chemical moiety having suitable chemical properties for
incorporation of a stable isotope, allowing the generation of
differentially labeled reagents which can be used to differentially
tag a polypeptide in two samples. The isotope tag, in combination
with the linker, has a sufficient length to allow the reactive
group to bind to a sample polypeptide and the affinity tag to bind
to its cognate binding partner. The isotope tag also has an
appropriate composition to allow incorporation of a stable isotope
at one or more atoms. A particularly useful stable isotope pair is
hydrogen and deuterium, which can be readily distinguished using
mass spectrometry as light and heavy forms, respectively. Any of a
number of isotopic atoms can be incorporated into the isotope tag
so long as the heavy and light forms can be distinguished using
mass spectrometry, for example, .sup.2H, .sup.13C, .sup.15N, 17O,
.sup.18O or .sup.34S. Differential isotopic tags will generally
differ by minimally 2 mass units and up to about 20 mass units, for
example, about 4 to about 16 mass units, about 6 to about 12 mass
units, and the like. Accordingly, the mass difference can be 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mass
units, or even a greater number of mass units. One skilled in the
art can readily determine an appropriate mass differential suitable
for isotope tags for detecting mass differences of VICAT-labeled
molecules using mass spectrometry. A multiple of a particular
isotope can be incorporated and/or a combination of isotopes (see
FIGS. 1 and 2).
[0050] Suitable criteria for selection of an affinity tag include
but are not limited to a desired size, solubility, the presence of
a suitable type and number of atoms for incorporation of desired
heavy isotope(s) sufficient for MS analysis, and the like. Any of
these or other desirable criteria can be considered by one skilled
in the art for affinity tag to be incorporated into a VICAT reagent
of the invention.
[0051] Exemplary isotope tags include the
4,7,10-trioxa-1,13-tridecanediam- ine based linker and its related
deuterated form, 2,2',3,3',11,11',12,12'--
octadeutero-4,7,10-trioxa-1,13-tridecanediamine, described by Gygi
et al. (supra, 1999). Other exemplary isotope tags include a
diethylenediamine, as shown in FIGS. 1, 2 and 4. One skilled in the
art can readily determine any of a number of appropriate isotope
tags useful in an VICAT-type affinity reagent that satisfy the
above-described criteria. As used herein, the portion of the VICAT
reagent which will remain attached to a sample molecule upon
cleavage of a cleavable linker and which has sufficient chemical
characteristics to allow the incorporation of isotopic atoms is
considered to be an isotope tag. It is understood that such a
portion is considered an isotope tag, regardless of whether
different isotopes have been incorporated into the tag. Thus, as
shown in FIGS. 1 and 2, three VICAT reagents are depicted that are
differentially labeled in the isotope tag, one with no heavy
isotope and the others depicted as +6 and +12 forms.
[0052] Thus, an isotope tag can be an alkyl, alkenyl, alkynyl,
alkoxy, aryl, and the like; at least one of the carbons can be
replaced with O, S, N; can contain O, S, NH, NR, NRR', CO, COO,
COS, S--S, SO, SO.sub.2, SH, CO--NR', CS--NR', Si--O, aryl or
diaryl group, where R is an alkyl, alkenyl, alkynyl, alkoxy, or
aryl group and R' is hydrogen, an alkyl, alkenyl, alkynyl, alkoxy
or aryl group; and can be optionally substituted, for example, with
O, S, N, and the like, and can contain an amine, carboxyl,
sulfhydryl, and the like (see WO 00/11208). An isotope tag is
generally of about 2 to about 100 atoms, although can be larger,
for example, about 2 to about 50 atoms, about 2 to about 40 atoms,
about 2 to about 30 atoms, about 2 to about 20 atoms, or about to 2
to about 10 atoms in length.
[0053] The above-described isotope tags and other derivatives can
be made in the same manner as that disclosed herein using methods
well known to those skilled in the art. One skilled in the art will
readily recognize that a number of suitable chemical groups can be
used as an isotope tag so long as the isotope tag can be
differentially isotopically labeled. One skilled in the art can
readily determine a number of suitable isotope tags of various
sizes based on criteria such as desired size of the tag, chemical
composition for incorporation of desired atom types and number of
differential isotopes, convenience of chemical synthesis, chemical
stability, and the like. The isotope tag has at least a minimal
size that allows sufficient differential labeling of the isotope
tag suitable for analysis in a mass spectrometer. For example, the
isotope tag is selected so that a sufficient mass differential can
be detected in a mass spectrometer, taking into account naturally
occurring isotope distribution (mostly .sup.13C) that can overlap
with the isotope tagged molecules if the mass differential is
small. One skilled in the art can readily recognize these and other
desirable characteristics of an isotope tag.
[0054] The use of an isotope tag is particularly useful for
quantifying the amount of sample molecules using mass spectrometry.
The method is based on derivatizing a specimen molecule such as a
polypeptide with a VICAT reagent. A control reference sample, for
example, synthesized known proteins or peptides or a control
sample, and a test sample are differentially labeled with lighter
and heavier forms of the VICAT affinity reagent. For quantitative
analysis, the derivatized samples can be combined and analyzed in
parallel using mass spectrometry, as described below (see Gygi et
al., supra, 1999).
[0055] A VICAT reagent of the invention also has a "reactive
group," which is reactive with a chemical moiety on a molecule in a
sample and therefore can be covalently coupled to a molecule in a
sample such as a polypeptide. Reactive groups are well known to
those skilled in the art (see, for example, Hermanson, supra, 1996;
Glazer et al., Laboratory Techniques in Biochemistry and Molecular
Biology: Chemical Modification of Proteins, Chapter 3, pp. 68-120,
Elsevier Biomedical Press, New York (1975); Pierce Catalog, Pierce,
Rockford Ill.). Any of a variety of reactive groups can be
incorporated into a VICAT type affinity reagent so long as the
reactive group can be covalently coupled to a polypeptide or other
desired molecule in a sample. The reactive group can also be a
group that reacts with a moiety on a target sample molecule in the
presence of an extrinsic reagent such as a crosslinking reagent.
For example, bis-maleimide can be used to crosslink an SH group on
a peptide with an SH group on the VICAT reagent. These and other
cross-linking reagents suitable for coupling a peptide to a VICAT
reagent are well know to those skilled in the art (see, for
example, Hermanson, supra, 1996; Glazer et al., supra, 1975;
Pierce).
[0056] Suitable criteria for selection of a reactive group include
but are not limited to the type of molecule to be targeted with the
VICAT reagent, the particular chemical moiety on the target
molecule to be reacted, the stability of the reactive group under
the buffer conditions used for the reaction, and the like. Any of
these or other desirable criteria can be considered by one skilled
in the art for affinity tag to be incorporated into a VICAT reagent
of the invention.
[0057] For example, a polypeptide can be coupled to the ICAT type
affinity reagent via a sulfhydryl reactive group, which can react
with free sulfhydryls of cysteine or reduced cystines in a
polypeptide. An exemplary sulfhydryl reactive group includes an
iodoacetamido group (see Gygi et al., supra, 1999). Other examplary
sulfhydryl reactive groups include maleimides, alkyl and aryl
halides, haloacetyls, .alpha.-haloacyls, pyridyl disulfides,
aziridines, acrylolyls, arylating agents and thiomethylsulfones. If
desired, the polypeptides can be reduced, for example, with
tri-butylphosphine, dithiothreitol, mercaptoethanol, and the like,
prior to reacting with a VICAT type reagent, which is particularly
useful when the VICAT reagent contains a sulfhydryl reactive
group.
[0058] A reactive group can also react with amines such as the
.alpha.-amino group of a peptide or the e-amino group of the side
chain of Lys, for example, imidoesters, N-hydroxysuccinimidyl
esters (NHS), isothiocyanates, isocyanates, acyl azides, sulfonyl
chlorides, aldehydes, ketones, glyoxals, epoxides (oxiranes),
carbonates, arylating agents, carbodiimides, anhydrides, and the
like. In addition, a vinyl sulfone such as
R--SO.sub.2--CH.dbd.CH.sub.2 or R--NHSO.sub.2--CH.dbd.CH.sub.2,
which can undergo a nucleophilic Michael-type addition reaction,
can be used to react with sulfhydryl or amine groups on the target
molecule.
[0059] A reactive group can also react with carboxyl groups found
in Asp or Glu or the C-terminus of a peptide, for example,
diazoalkanes, diazoacetyls, carbonyldiimidazole, carbodiimides, and
the like. A reactive group that reacts with a hydroxyl group
includes, for example, epoxides (oxiranes), carbonyldiimidazoles,
N,N'-disuccinimidyl carbonates, N-hydroxycuccinimidyl
chloroformates, and the like. A reactive group can also react with
amino acids such as His, Tyr, Arg, and Met. In addition, a reactive
group can also react with a phosphate group for selective labeling
of phosphopeptides, or with other covalently modified peptides,
including glycopeptides, lipopeptides, or any of the known covalent
polypeptide modifications. One skilled in the art can readily
determine conditions for modifying sample molecules by using
various reagents, incubation conditions and time of incubation to
obtain conditions optimal for modification of sample molecule for
use in reagents and methods of the invention.
[0060] The reactive groups described above can form a covalent bond
with the target sample molecule. However, it is understood that a
VICAT reagent can contain a group that can non-covalently interact
with a sample molecule so long as the interaction has high
specificity and affinity.
[0061] A VICAT reagent of the invention can contain any
combinations of the above-described affinity tags, detectable
moieties, linkers, isotope tags, and reactive groups, as well as
others well known to those skilled in the art. For example, a VICAT
reagent can contain the affinity tag biotin, the detectable moiety
rhodamine, the photolabile linker 2-nitrobenzyloxycarbonyl, the
isotope tag ethylenediamine, and the reactive group iodacetyl, as
depicted in FIG. 2. Another VICAT reagent can contain the affinity
tag biotin, the detectable moiety 7-nitrobenz-2-oxa-1,3-diazole
(NBD), the photolabile linker 2-nitrobenzyloxycarbonyl, the isotope
tag ethylenediamine, and the reactive group iodacetyl, as depicted
in FIG. 1. The reagent depicted in FIG. 1 is particularly useful
because it is easy to make, is stable, and has solubility
properties in aqueous solution sufficient for quantitative
proteomics analysis. An additional VICAT reagent can contain the
affinity tag biotin, the radioactive detectable moiety
.sup.14C-acetyl, the isotope tag ethylenediamine, and the reactive
group iodacetyl, as depicted in FIG. 4.
[0062] Still another VICAT reagent can contain the affinity tag
glutathione, the detectable moiety coumarin, a linker containing a
benzylsulfonyl group, the isotope tag tridecanediamine, and an
isothiocyanate reactive group. Yet another VICAT reagent can
contain the affinity tag poly-His with six histidines, the
detectable moiety BIDIPY, a linker containing a disulfide, the
isotope tag dodecane, and a disuccinimidyl carbonate reactive
group.
[0063] The above-described VICAT reagents are merely exemplary of
the VICAT reagents that can be readily prepared by one skilled in
the art in the same manner as that disclosed herein using well
known methods of chemical synthesis, including methods similar to
those exemplified herein (see Example I). Based on the desired
application of the VICAT reagent, one skilled in the art can select
a suitable affinity tag, detectable moiety, linker, isotope tag,
and reactive group sufficient for desired criteria, as disclosed
herein.
[0064] The VICAT reagents of the invention can be used to label
molecules in a sample. The methods are particularly useful for
labeling polypeptides in a sample, and can thus be used for
qualitative and quantitative proteome analysis. Although labeling
of polypeptides is exemplified herein, it is understood that sample
molecule having chemical properties reactive with the VICAT reagent
reactive group can be labeled with a VICAT reagent of the
invention. For example, a VICAT reagent can be used to label
nucleic acids, carbohydrates, lipids, metabolites or other sample
molecules.
[0065] The invention also provides a method of isolating a
polypeptide in a sample. The method can include the steps of
contacting a sample with a VICAT reagent of the invention under
conditions allowing the reactive group to bind to and react with
one or more polypeptides in the sample, thereby tagging one or more
polypeptides with the reagent; resolving the polypeptides in the
sample; visualizing the polypeptides tagged with the reagent;
contacting the tagged polypeptides with a capture moiety for the
affinity tag; and isolating the tagged polypeptides. It is
understood that the steps of this method and other methods of the
invention can be performed in any order so long as a VICAT reagent
tags a sample molecule and can be used to isolate a target
molecule. For example, an affinity isolation step of a
VICAT-labeled molecule can be performed before or after other
fractionation steps used to resolve molecules in a sample and can
be performed before or after a visualization step. Furthermore, a
sample can be resolved using various fractionation methods prior to
labeling with a VICAT reagent. One skilled in the art can readily
determine an appropriate order for carrying out steps of the
methods of the invention suitable for tagging and isolating a
sample molecule.
[0066] As used herein, the term "polypeptide" refers to a peptide
or polypeptide of two or more amino acids. A polypeptide can also
be modified by naturally occurring modifications such as
post-translational modifications, including phosphorylation, fatty
acylation, prenylation, sulfation, hydroxylation, acetylation,
addition of carbohydrate, addition of prosthetic groups or
cofactors, formation of disulfide bonds, proteolysis, assembly into
macromolecular complexes, and the like.
[0067] The invention provides a method of labeling a polypeptide in
a sample by contacting a sample with a VICAT reagent of the
invention under conditions allowing the reactive group to bind to
one or more polypeptides in the sample. One skilled in the art can
readily determine appropriate conditions so that the reactive group
of a VICAT reagent can react with a sample molecule, including
appropriate buffers, salts, pH, temperature, and the like (see
Hermanson, supra, 1996). Thus, a VICAT reagent of the invention can
be used to label a sample molecule via the reactive group,
resulting in the attachment of the VICAT reagent and corresponding
isotope tag to a sample molecule.
[0068] Once a VICAT reagent has reacted with a sample molecule, the
sample is labeled in a manner that allows convenient visualization
of the labeled molecule(s). For example, any of the visualization
methods disclosed herein can be used to conveniently locate the
labeled molecules. Such a visualization is particularly.useful for
locating a labeled sample molecule when a complex sample molecule
has been resolved from other sample molecules. As used herein,
"resolving" when used in reference to a sample molecule refers to
using a purification or fractionation step that separates the
sample molecule from at least one other molecule in the sample.
Thus, the method can further include the step of visualizing the
molecules tagged with the VICAT reagent.
[0069] As used herein, the term "sample" is intended to mean any
biological fluid, cell, tissue, organ or portion thereof, that
includes one or more different molecules such as nucleic acids,
polypeptides, or small molecules. A sample can be a tissue section
obtained by biopsy, or cells that are placed in or adapted to
tissue culture. A sample can also be a biological fluid specimen
such as blood, urine or saliva. A sample can additionally be a cell
extract from any species, including prokaryotic and eukaryotic
cells as well as viruses. A tissue sample can be further
fractionated, if desired, to a fraction containing particular cell
types.
[0070] Furthermore, the sample can be fractionated by a number of
known fractionation techniques. Since such fractionation methods
separate molecules, such techniques are used to resolve sample
molecules. Methods for resolving sample molecules are well known to
those skilled in the art. Fractionation methods, which can be used
to resolve sample molecules, include but are not limited to
subcellular fractionation or chromatographic techniques such as ion
exchange, including strong and weak anion and cation exchange
chromatography, hydrophobic and reverse phase, size exclusion,
affinity, hydrophobic charge-induction chromatography, dye-binding,
and the like (Ausubel et al., Current Protocols in Molecular
Biology (Supplement 56), John Wiley & Sons, New York (2001);
Scopes, Protein Purification: Principles and Practice, third
edition, Springer-Verlag, New York (1993); Burton and Harding, J.
Chromatogr. A 814:71-81 (1998)). Other fractionation methods
include, for example, centrifugation, electrophoresis, the use of
salts, and the like (see Scopes, supra, 1993).
[0071] Affinity chromatography can also be used including, for
example, dye-binding resins such as Cibacron blue, substrate
analogs, including analogs of cofactors such as ATP, NAD, and the
like, ligands, specific antibodies useful for immuno-affinity
isolation, either polyclonal or monoclonal, and the like. An
exemplary affinity resin includes affinity resins that bind to
specific moieties that can be incorporated into a polypeptide such
as an avidin resin that binds to a biotin tag on a sample molecule
labeled with a VICAT reagent of the invention, as disclosed herein.
The resolution and capacity of particular chromatographic media are
known in the art and can be determined by those skilled in the art.
The usefulness of a particular chromatographic separation for a
particular application can similarly be assessed by those skilled
in the art.
[0072] Those of skill in the art will be able to determine the
appropriate chromatography conditions for a particular sample size
or composition and will know how to obtain reproducible results for
chromatographic separations under defined buffer, column dimension,
and flow rate conditions. The fractionation methods can optionally
include the use of an internal standard for assessing the
reproducibility of a particular chromatographic application or
other fractionation method. Appropriate internal standards will
vary depending on the chromatographic medium or the fractionation
method used. Those skilled in the art will be able to determine an
internal standard applicable to a method of fractionation such as
chromatography.
[0073] Electrophoresis, including gel electrophoresis or capillary
electrophoresis, can also be used to resolve sample molecules. As
disclosed herein, isoelectric focusing (IEF) is a particularly
useful method to resolve sample polypeptides. As discussed herein,
sample molecules can be processed, for example, by protease
cleavage into peptide fragments. Accordingly, when referring to
sample molecules, the sample molecules can be intact as found in an
original sample or can be processed, for example, into smaller
molecules such as peptides from a polypeptide sample.
[0074] If desired, sample molecules can be modified, either before
or after a fractionation step. For example, the methods of the
invention are particularly useful for mass spectrometry (MS)
analysis. In the case of MS analysis of polypeptides, it is often
useful to cleave the polypeptide into smaller fragments, for
example, by proteolysis. Thus, a polypeptide molecule can be
enzymatically cleaved with one or more proteases into peptide
fragments. Exemplary proteases useful for cleaving polypeptides
include trypsin, chymotrypsin, pepsin, papain, Staphylococcus
aureus (V8) protease, and the like. Polypeptides can also be
cleaved chemically, for example, using CNBr, acid or other chemical
reagents.
[0075] For polypeptide fragmentation, the polypeptides in the
sample mixture, or the polypeptides contained in each fraction if
optional sample fractionation is employed, can be subjected to
sequence specific cleavage, such as cleavage by trypsin. The use of
sequence specific cleavage can be particularly useful because the
termini of peptides cleaved by a sequence specific method can act
as a constraint. However, it is understood that the cleavage method
used to generate fragments need not be sequence specific, if
desired.
[0076] Furthermore, for polypeptide tagging, the polypeptides in
the sample can be denatured and optionally reduced. Reducing the
sample can be particularly useful when the reactive group on the
VICAT reagent is reactive with a thiol. Other useful reactive
groups include amino or carboxyl groups of polypeptides or specific
post-translational modifications, including phosphate, carbohydrate
or lipid.
[0077] The methods of the invention involve the coupling of a
VICAT-type reagent to a sample molecule, which results in the
incorporation of a visible tag, which is detectable as disclosed
herein. Such a visible tag is particularly useful for visualizing
the location of a sample molecule resolved by a fractionation
method. Thus, in a complex sample, the visible tag allows the
convenient localization of a VICAT-labeled molecule. Furthermore,
as disclosed herein, a specific sample molecule can be identified
by preparing known standards and running the standards in parallel
or in the same sample to facilitate the visualization of a
particular desired sample molecule. If the standard molecules are
added to the sample molecules, a relatively high amount of the
standard can be added to indicate the position of the corresponding
target molecule of interest.
[0078] The ability to readily visualize a VICAT-labeled molecule
facilitates further characterization of the labeled molecule,
regardless of the complexity of the original sample. In a complex
sample, it is likely that a number of sample molecules will be
labeled by the reactive group of the VICAT reagent. For example,
the methods of the invention are particularly useful for proteomics
analysis, where the sample molecules to be analyzed are
polypeptides or peptide fragments thereof. By running a sample in
parallel with a known standard, a particular sample molecule can be
readily visualized. A known sample molecule can be synthesized,
labeled with a VICAT reagent, and run in parallel under essentially
the same conditions as the sample molecules. For example, a known
polypeptide can be synthesized, optionally added to an unlabeled
sample, and resolved in the same manner as the VICAT-labeled
sample. Since the samples molecules are run under similar
conditions, the location of the known polypeptide, also tagged with
the VICAT reagent, can readily be determined in the parallel
sample. Alternatively, a known molecule can be synthesized and
labeled with the same VICAT reagent, but having a different
detectable moiety, and run together with the sample molecules. In
such a case, the different detectable moiety would have essentially
the same resolution as the other detectable moiety in the
particular fractionation method used. An example of this is the use
of a radiolabel as the detectable moiety. The radiolabeled standard
is added to the sample, and the position of the analyte to be
analyzed is detected by autoradiography, fluorimetry, phospor
storage imaging, or liquid scintillation or Cerenkov counting.
[0079] Furthermore, a VICAT-labeled standard molecule can be used
to calibrate a particular separation method so that the position of
a corresponding VICAT-labeled sample molecule can be predicted when
run under substantially similar conditions. Because a separation
method can be performed reproducibly when run under substantially
similar conditions, a VICAT-labeled standard molecule can be run on
a separation medium under defined conditions so that the location
of the VICAT-labeled standard molecule can be used to predict the
location of a VICAT-labeled sample molecule run under the same
defined conditions. This obviates the need to add the standard
molecule to the sample or to run the standard molecule in parallel.
Such an approach can be particularly useful for analysis by
IEF.
[0080] Once a VICAT-labeled molecule of interest has been
visualized and located in the separation medium, the VICAT-labeled
molecule can be isolated by contacting with a capture moiety
corresponding to the affinity tag of the VICAT reagent.
Alternatively, the VICAT-labeled molecule can be affinity isolated
by the capture moiety prior to resolution on a separation medium or
visualization. For example, if the sample molecules are resolved by
IEF electrophoresis, the region containing the VICAT-labeled sample
molecule of interest can be extracted and contacted with an
appropriate capture moiety (see Example III). Thus, by visualizing
the resolved sample molecules to locate a VICAT-labeled molecule,
the VICAT-labeled molecule of interest can be further processed and
affinity purified in the absence of other VICAT-labeled
molecules.
[0081] The capture moiety can be attached, for example, to a bead,
resin, membrane or disk, or can be coupled to any type of solid
support suitable for the isolation of the VICAT-labeled
molecule(s). It is understood that the affinity tag can bind to a
capture moiety directly bound to the solid support or to a capture
moiety that binds via a secondary agent to the solid support. For
example, the capture moiety can be bound in solution to the
VICAT-labeled molecule and subsequently bound to a solid support,
such as by binding to an antibody specific for the capture moiety.
One skilled in the art can readily determine a desired method to
affinity isolate a VICAT-labeled molecule using well known affinity
isolation methods.
[0082] The invention thus provides a method of isolating one or
more polypeptides in a sample. The method can include the steps of
contacting a sample with a VICAT reagent of the invention under
conditions allowing the reactive group to bind to and react with
one or more polypeptides in sample, thereby tagging one or more
polypeptides with the reagent; resolving the polypeptides in the
sample; visualizing the polypeptides tagged with the reagent;
contacting the tagged polypeptides with a capture moiety for the
affinity tag; and isolating the tagged polypeptides. In addition to
labeling polypeptides, this and other methods of the invention can
be used to label other types of sample molecules such as
carbohydrates, nucleic acids, and the like, if desired. Since the
linker of the VICAT reagent can be cleavable, the method can
further include the step of cleaving the linker. The cleavage
results in the release of the VICAT-labeled molecule such that the
molecule retains the isotope tag but is separated from the
detectable moiety and affinity tag. Thus, the VICAT-labeled
molecules can be readily released from an affinity resin or other
solid support to which the labeled molecules are bound.
[0083] The isolated VICAT-labeled molecules can be further
characterized, if desired. A particularly useful method for
characterizing sample molecules is mass spectrometry (MS), which
can be used to identify and/or quantify the VICAT-labeled molecule.
Thus, the invention provides a method of identifying a polypeptide
in a sample. The method can include the steps of contacting a
sample with a VICAT reagent of the invention under conditions
allowing the reactive group to bind to and react with one or more
polypeptides in the sample, thereby tagging one or more
polypeptides with the reagent; resolving the polypeptides in the
sample; visualizing the one or more polypeptides tagged with the
reagent; contacting the one or more tagged polypeptides with a
capture moiety for the affinity tag; isolating the one or more
tagged polypeptides; cleaving the linker of the reagent to release
the one or more tagged polypeptides; and identifying a released
tagged polypeptide.
[0084] The invention additionally provides a method of identifying
a polypeptide in a sample. The method can include the steps of
contacting a sample with a VICAT reagent of the invention under
conditions allowing the reactive group to bind to and react with
one or more polypeptides in the sample, thereby labeling one or
more polypeptides with the reagent; cleaving the polypeptides in
the sample to generate peptide fragments; adding one or more
peptide standards to the sample, wherein the peptide standards
correspond to peptides generated from cleaving sample polypeptides
and are labeled with an isotopically distinct version of the
isotope tag; resolving the labeled peptides in the sample;
visualizing the labeled peptides; contacting the labeled peptides
with a capture moiety for the affinity tag; isolating the labeled
peptides; cleaving the linker of the reagent to release the
isolated peptides; and identifying a released peptide. The
identifying and/or quantifying step can be performed using mass
spectrometry or tandem mass spectrometry.
[0085] A variety of mass spectrometry systems can be employed in
the methods of the invention for identifying and/or quantifying a
sample molecule such as a polypeptide. Mass analyzers with high
mass accuracy, high sensitivity and high resolution include, but
are not limited to, matrix-assisted laser desorption time-of-flight
(MALDI-TOF) mass spectrometers, electrospray ionization
time-of-flight (ESI-TOF) mass spectrometers and Fourier transform
ion cyclotron mass analyzers (FT-ICR-MS). Other modes of MS include
ion trap and triple quadrupole mass spectrometers. In ion trap MS,
analytes are ionized by electrospray ionization or MALDI and then
put into an ion trap. Trapped ions can then be separately analyzed
by MS upon selective release from the ion trap. Fragments can also
be generated in the ion trap and analyzed. Sample molecules such as
polypeptides labeled with a VICAT type reagent can be analyzed, for
example, by single stage mass spectrometry with a MALDI-TOF or
ESI-TOF system. Methods of mass spectrometry analysis are well
known to those skilled in the art (see, for example, Yates, J. Mass
Spect. 33:1-19 (1998); Kinter and Sherman, Protein Sequencing and
Identification Using Tandem Mass Spectrometry, John Wiley &
Sons, New York (2000); Aebersold and Goodlett,. Chem. Rev.
101:269-295 (2001); Griffin et al., Curr. Opin. Biotechnol.
12:607-612 (2001)).
[0086] For high resolution polypeptide fragment separation, liquid
chromatography ESI-MS/MS or automated LC-MS/MS, which utilizes
capillary reverse phase chromatography as the separation method,
can be used (Yates et al., Methods Mol. Biol. 112:553-569 (1999)).
Data dependent collision-induced dissociation (CID) with dynamic
exclusion can also be used as the mass spectrometric
method(Goodlett, et al., Anal. Chem. 72:1112-1118 (2000)).
[0087] Differentially labeled isotope tags can be incorporated into
the VICAT reagent (see Example I). The differential tags can be
reacted with a test and control sample and run together or in
parallel. The differentially labeled molecules can be analyzed
using mass spectrometry (MS). Because the sample molecules are
differentially labeled with light and heavy isotope tags, the
peptide fragments can be distinguished by MS, allowing a
side-by-side comparison of the relative amounts of each peptide
fragment from a control sample and a test sample. If desired, MS
can also be used to sequence the corresponding labeled peptides,
allowing identification of molecules corresponding to the tagged
peptide fragments.
[0088] Moreover, the use of an isotope tag in the VICAT reagent
allows the incorporation of various heavy labels into the same
molecule. A VICAT reagent having 0, +6 or +12 heavy isotopes is
exemplified in FIGS. 1 and 2. By having chemically identical but
differentially isotopically labeled VICAT reagents available,
multiple tags can be used in the same experiment. For example, one
VICAT reagent can be used to tag sample molecules, a second VICAT
reagent can be used to tag a standard molecule for use as an
internal standard, and a third VICAT reagent can be used as a
marker for the sample molecule in a separation method. Thus, the
ability to incorporate various amounts. of heavy isotope into an
isotope tag provides versatility in the use of the VICAT reagent
for labeling and isolating sample molecules, preparing standards,
and visualizing sample molecules for isolation and analysis.
[0089] The use of multiple isotopic forms of the isotope tag of a
VICAT reagent is advantageous for allowing tagging of a sample
molecule, convenient localization of a sample molecule in a
separation method, and quantification of the sample molecule by
inclusion of an internal standard. As discussed above, a
VICAT-labeled standard peptide can be added at relatively high
quantities to facilitate localization in a separation method. The
sample molecule can be labeled with the same VICAT reagent but with
a differential tag, for example, a +6 tag. Furthermore, an internal
standard labeled with still another differential tag, for example
+12, can be included for quantification. In this case, one
VICAT-labeled standard is added at a high concentration for
visualization while a second VICAT-labeled standard is added in an
amount suitable for quantifying the sample molecule. The internal
standard added for quantifying the sample molecule is added in an
amount suitable for quantification, that is, within the
quantitative range of the detection method. One skilled in the art
can readily determine an appropriate amount of internal standard to
add to a sample for quantitative analysis and adjust the amount
added depending on the type of quantitative analysis used. For
example, the amount of internal standard can be adjusted so that
the VICAT-labeled sample and standard molecules can be quantified
by mass spectrometry. Similarly, one skilled in the art can readily
determine a suitable amount of VICAT-labeled standard to use for
visualization in a separation method depending on the nature of the
detectable moiety on the VICAT reagent and the detection method
used.
[0090] An advantage of the VICAT method is that a pair of peptides
tagged with light and heavy VICAT reagents are chemically identical
and therefore serve as mutual internal standards for accurate
quantification (Gygi et al., supra, 1999). Using MS, the ratios
between the intensities of the lower and upper mass components of
pairs of heavy- and light-tagged fragments provides an accurate
measure of the relative abundance of the peptide fragments.
Furthermore, a short sequence of contiguous amino acids, for
example, 5-25 residues, contains sufficient information to identify
the unique polypeptide from which the peptide fragment was derived
(Gygi et al., supra, 1999). Thus, the VICAT method can be
conveniently used to identify differentially expressed molecules,
if desired.
[0091] Furthermore, the methods of the invention are advantageous
in that absolute quantification of a sample molecule can be
determined if a known amount of VICAT-labeled standard molecule is
included as a standard for quantitative analytical methods. The
inclusion of a detectable tag in a VICAT reagent can be readily
exploited to facilitate quantification and calibration of a
VICAT-labeled standard. If a VICAT reagent contains a detectable
moiety that absorbs light, the detectable moiety can be used to
quantify a labeled molecule based on the absorption properties of
the detectable moiety. Exemplary detectable moieties that absorb
light include fluorophores such as coumarin. For example, if the
detectable moiety absorbs in the ultraviolet (UV) range, UV
absorbance can be used to determine the concentration of the
labeled standard peptide. The concentration can be determined from
Beer's law and the known extinction coefficient of the detectable
moiety used in the VICAT reagent.
[0092] As discussed above, an isotope tag is included in a VICAT
reagent, allowing differential labels to be incorporated into
sample and standard molecules and facilitating quantification of
sample molecules. Thus, a VICAT reagent can be used for two
purposes, localization and quantification. The VICAT reagent is
used to determine the location of a particular sample molecule
based on visualization of the corresponding VICAT-labeled standard.
The VICAT reagent also allows quantification by MS analysis based
on the incorporation of isotopically distinct isotope tags, which
can be used for quantitative comparison of the VICAT-labeled sample
and standard molecules.
[0093] Although generally exemplified herein with the addition a
standard peptide or peptides labeled with a corresponding
isotopically labeled VICAT reagent for analysis of a particular
sample polypeptide, it is understood that the methods of the
invention are also applicable to the analysis of multiple sample
polypeptides. For example, VICAT-labeled standard peptides can be
added to identify and/or quantify two different polypeptides in a
sample. In such a case, a particular VICAT reagent can be used to
label sample polypeptides, and the corresponding standard peptides
for the two polypeptides are labeled with an isotopically distinct
version of the same VICAT reagent as used to label the sample
polypeptides. The methods are advantageous in that multiple
polypeptides in a sample can be analyzed.
[0094] Any number of different polypeptides in a sample can be
analyzed so long as there is sufficient resolution of the standard
VICAT-labeled peptides for further analysis. Thus, the methods of
the invention can be used to identify and/or quantify three or more
polypeptides in a sample, four or more polypeptides, five or more
polypeptides, six or more polypeptides, seven or more polypeptides,
eight or more polypeptides, nine or more polypeptides, ten or more
polypeptides, twelve or more polypeptides, fifteen or more
polypeptides, or even grater numbers so long as the VICAT-labeled
peptides can be sufficiently resolved for further analysis.
Corresponding VICAT-labeled standards for the multiple polypeptides
to be analyzed can be synthesized and added to the sample.
[0095] When analyzing multiple polypeptides in a sample, a single
VICAT reagent can be used to label sample polypeptides. In
addition, more than one type of VICAT reagent can be used to label
sample polypeptides. For example, one type of VICAT reagent can be
used to label reactive sulfhydryl groups of sample polypeptides and
a second type of VICAT reagent can be used to label reactive amine
groups. The use of VICAT reagents having different reactive groups
to label the same sample can be used to distinguish between two
polypeptides. Furthermore, such VICAT reagents can also incorporate
different detectable moieties, for example, two different
fluorophores or chromophores. Thus, the use of different types of
VICAT reagents containing different reactive groups and/or
detectable moieties can be used to facilitate the identification
and quantification of multiple polypeptides in a sample. One
skilled in the art can readily determine suitable VICAT reagents
for differential labeling of two or more polypeptides in a
sample.
[0096] The methods of the invention can be used in a variety of
applications. The methods of the invention are particularly useful
for analysis of polypeptides for which antibodies are not
available. For example, the methods can be used to analyze
polypeptides for which antibodies have not been generated or which
are poorly immunogenic. In the case where new gene sequences or
splice variants are identified, there is no need to recombinantly
express the encoded polypeptide for antibody production. Instead,
only one or a few peptides that uniquely identify a polypeptide
need to be synthesized, which can be readily predicted from the
nucleic acid sequence.
[0097] The methods of the invention can also be applied to the
quantification of specific isoforms of splice variants. The
peptides selected as standards are chosen based on known or
empirically determined splice variants. The peptide standards are
selected such that, for each alternatively spliced protein,
minimally one peptide is used that is common to all splice variants
and minimally one peptide is used that is unique for each splice
variant tested.
[0098] The methods can also be applied to the detection and
quantification of proteins associated with genetic disease. As
such, a genetic disease known to be associated with aberrant
expression of a polypeptide can be analyzed using methods of the
invention by including a VICAT-labeled peptide standard
corresponding to the aberrantly expressed polypeptide.
[0099] An exemplary genetic disease for which methods of the
invention can be applied is muscular dystrophy (see Example IV).
Duchenne and Becker muscular dystrophies are allelic disorders
caused by a deficiency of the dystrophin protein in muscle (Worton
and Brooke, in The Metabolic and Molecular Bases of Inherited
Diseases, 7th ed., Scriver, ed., ppl 4195-4112, McGraw-Hill, New
York (1994)). The dystrophin gene, located on the X chromosome at
Xp21, is one of the largest genes in the human genome (2,300 kb),
contains 79 exons, and encodes a 427 kDa cytoskeletal protein. The
dystrophin protein is missing in boys with Duchenne muscular
dystrophy (DMD) and is significantly decreased in those with Becker
muscular dystrophy (BMD).
[0100] From a diagnostic perspective, approximately 2/3 of affected
males can be confirmed to have muscular dystrophy by molecular
methods that can identify deletions or duplications within the
dystrophin gene (Chamberlain et al., Nucl. Acids Res.
16:11141-11156 (1988)). Confirmatory diagnosis of DMD or BMD in the
remaining 1/3 of patients who have small genetic alterations
typically involves a surgical muscle biopsy with antibody staining
or immunoblot analysis to show the absence of dystrophin. A
truncated dystrophin revealed by immunoblot analysis, or an
in-frame deletion in the central portion of the gene, would predict
the Becker form of muscular dystrophy. The average cost associated
with an open muscle biopsy and pathological evaluation exceeds
$3000 and places children with muscular dystrophy at clinical risk
from anesthesia, with the potential for rhabdomyolosis occurring
from the anesthesia.
[0101] A VICAT reagent of the invention can be applied to
quantitatively measure dystrophin from very small muscle samples
that can be obtained by needle biopsy. Such methods are also
applicable to other forms of muscular dystrophy. Thus, the VICAT
reagents of the invention can be used in methods directed to
microtechnology for the quantitative measurement of specific
proteins in muscle tissue. In addition to diagnostic applications,
the methods can also be applied to measuring the effectiveness of
gene therapy or other therapies used to treat a disease.
[0102] The reagents and methods of the invention are also
applicable to diagnosing and determining the prognosis for various
cancers. For example, mRNA expression array experiments are
performed on tumor samples of various tissues, for example, lung,
colon, liver, ovary, breast, prostate, and the like. The mRNA
expression experiments allow the determination of tumor-associated
changes in gene expression, either up-regulation or
down-regulation, compared to normal corresponding tissue.
Furthermore, it is useful to determine whether any of the
abberantly expressed proteins, particularly those having increased
expression, are present in blood serum of the patients. If so,
these up-regulated proteins can function as convenient markers for
diagnosis of the corresponding cancer in serum samples from
patients. Exemplary cancer diagnostic markers include prostate
specific antigen (PSA) for prostate cancer and CA125 for ovarian
cancer. The VICAT reagents and methods of the invention are
particularly useful for rapidly screening "candidate" diagnostic
markers by measuring the level of the corresponding protein in
serum or other bodily fluids and determining whether the amount of
candidate diagnostic markers correlate with the disease.
[0103] Thus, the invention provides a method of diagnosing a
disease. The method can include the steps of contacting a sample
from a patient with a VICAT reagent of the invention under
conditions allowing the reactive group to bind to and react with
one or more polypeptides in the sample, thereby labeling one or
more polypeptides with the reagent; cleaving the polypeptides in
the sample to generate peptide fragments; adding one or more
peptide standards to the sample, wherein the peptide standards
correspond to peptides generated from cleaving sample polypeptides,
are labeled with an isotopically distinct version of the isotope
tag, and are derived from a polypeptide exhibiting aberrant
expression in the disease; resolving the labeled peptides in the
sample; visualizing the labeled peptides; contacting the labeled
peptides with a capture moiety for the affinity tag; isolating the
labeled peptides; cleaving the linker of the reagent to release the
isolated peptides; quantifying the released peptides; and comparing
the amount of sample peptide to standard peptide in the released
peptides, wherein the amount of sample peptide is correlated with
the diagnosis of the disease. The methods can be used to diagnose a
variety of diseases in which aberrant protein expression is
associated with a disease, either increased or decreased
expression.
[0104] The methods of the invention are particularly useful for
diagnosis of genetic diseases, where one or a few aberrantly
expressed proteins are diagnostic for a disease. For example, the
methods can be used to diagnose a disease such as muscular
dystrophy, including Duchenne muscular dystrophy or Becker muscular
dystrophy by measuring decreased expression of dystrophin.
Similarly, the methods of the invention can be used to quantify the
expression of prostate specific antigen (PSA) for diagnosis of
prostate cancer. One skilled in the art will readily recognize that
the methods of the invention are applicable to these and other
diseases associated with aberrant expression of one or more
polypeptides. Furthermore, one skilled in the art can readily
generate corresponding standard peptides for analysis of the
aberrantly expressed polypeptides using methods of the
invention.
[0105] Thus, one skilled in the art can readily apply the methods
of the invention to the analysis of known markers associated with a
disease. An increasing number of protein markers for a variety of
diseases are becoming available through genomics and proteomics
analysis. For example, mRNA expression arrays can be used to
determine differential expression between a healthy individual and
an individual having a disease. VICAT-labeled standards
corresponding to the differentially expressed mRNAs can be
synthesized and used for qualitative and/or quantitative diagnostic
analysis of patient samples of the same tissue. Furthermore, if the
differentially expressed mRNAs encode one or more secreted proteins
or peptides, the methods of the invention can be applied to blood
or serum samples to analyze differentially expressed secreted
proteins correlated with a disease state.
[0106] The methods of the invention are also applicable to the
detection and quantification of patterns of protein expression that
are diagnostic or prognostic for a disease. The methods can be used
in diagnostic applications in body fluids such as blood, serum or
plasma, cerebrospinal fluid, urine, saliva, seminal plasma,
pancreatic juice, breast milk, lung lavage, and the like. Because
the methods can be used to analyze and quantify multiple peptides
in the same analysis, the methods are particularly useful for
diagnostic applications since the ability to analyze multiple
polypeptides in the same analysis allows multiple peptides
correlated with a diagnostic application to be used rather than the
analysis of one diagnostic marker alone. Thus, the methods can be
applied to the analysis of multiple diagnostic markers associated
with a disease. Once a set of diagnostic markers has been
identified, the methods of the invention can be applied to quantify
the set of markers for an analysis that is expected to be more
informative than the analysis of a single diagnostic marker
alone.
[0107] Diagnostic patterns of protein expression can be determined
by direct comparative, quantitative protein profiling of tissues or
body fluids of persons afflicted with a particular disease and
healthy control individuals (Gygi et al., Nat. Biotechnol.
17:994-999 (1999); WO 00/11208). Alternatively, diagnostic protein
expression patterns can be determined from gene expression array
measurements in afflicted tissue of an individual having a disease
and healthy control tissue of the same type. In addition, specific
marker proteins can be identified from known disease markers found
in the scientific literature. One skilled in the art will recognize
that these and other methods for determining or identifying
differential protein expression correlated with a disease state can
be used to identify protein disease markers suitable for diagnostic
applications using VICAT reagents and methods of the invention.
[0108] The invention additionally provides a kit containing one or
more VICAT reagents. For example, the kit can contain a single
VICAT reagent useful for labeling and identifying a polypeptide. In
addition, a kit can contain a set of two or more differentially
isotopically labeled VICAT reagents, which is particularly useful
for quantitative analysis using mass spectrometry. The contents of
the kit of the invention, for example, one or more VICAT reagents,
are contained in packaging material, and, if desired, a sterile,
contaminant-free environment. In addition, the packaging material
contains instructions indicating how the materials within the kit
can be employed to label sample molecules. The instructions for use
typically include a tangible expression describing the reagent
concentration or at least one assay method parameter, such as the
relative amounts of reagent and sample to be admixed, maintenance
time periods for reagent/sample admixtures, temperature, buffer
conditions, and the like.
[0109] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also provided within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
EXAMPLE I
[0110] Chemical Structure and Synthesis of VICAT Reagents
[0111] This example describes a synthetic scheme for VICAT
reagents.
[0112] The structure of exemplary VICAT reagents and their
corresponding synthetic schemes are-shown in FIGS. 1, 2 and 4. A
representative reagent contains the biotin and iodoacetyl features
that are present in the ICAT reagents (Gygi et al., Nature
Biotechnol. 17:994-999 (1999) and also contains a
tetramethylrhodamine to make the reagent highly colored and
fluorescent. The iodoacetyl group is attached to an ethylenediamine
unit, which is attached to the rest of the VICAT reagent via a
photolabile 2-nitrobenzyloxycarbonyl group. Exposure of the
VICAT-peptide conjugate to long wavelength UV light (using a
handheld lamp) for several minutes leads to photocleavage of the
bond between the benzyl carbon and the bridging ester oxygen,
yielding Peptide-S--CH.sub.2CONHCH.sub.2CH.sub.2NH- --COOH. The
latter undergoes spontaneous decarboxylation (Olejnik et al., Proc.
Natl. Acad. Sci. USA 92:7590-7594 (1995) to yield
Peptide-S--CH.sub.2CONHCH.sub.2CH.sub.2NH.sub.2. This peptide
cysteine-bound N-aminoethylacetamido group is abbreviated
NAMETAG.
[0113] Also shown in FIGS. 1 and 2 are the VICAT(+6) and VICAT(+12)
reagents, which contain heavy isotopes in the NAMETAG portion, thus
increasing their mass by +6 and +12, respectively. After
photocleavage, the NAMETAG group, with or without isotopic
substitution, is left on the peptide so that the peptides can be
quantified by the ICAT-based method (Gygi et al., supra, 1999).
[0114] Three different peptides, 10-15 amino acids in length and
containing a single NAMETAG-derivatized cysteine, were found to
undergo normal peptide bond fragmentation during tandem ESI-MS.
Thus, the presence of the NAMETAG does not interfere with obtaining
peptide sequence information. Additionally, the amide in the
NAMETAG moiety eliminates ethylenediamine upon collision-induced
dissociation (CID) ' which serves to fingerprint by MS/MS those
peptides that contain the NAMETAG. The VICAT reagents also have the
advantageous property of being water soluble at the concentrations
needed for peptide mixture analysis. In particular, the presence of
the tertiary amine in the linker will be protonated at pH near
neutrality or lower and thus contribute to reagent solubility in
water.
[0115] The NBD-VICAT reagent shown in FIG. 1A was synthesized as
shown in FIG. 1B. Briefly, tetrahydrofuran (THF) was distilled from
sodium metal/benzophenone ketyl. Methylene Chloride
(CH.sub.2Cl.sub.2), and triethylamine (TEA) were distilled from
CaH.sub.2. Anhydrous dimethylformamide (DMF) and pyridine were
purchased from Aldrich Chemical Co. (Milwaukee, Wis.). HPLC grade
solvents were purchase from Fisher (Fair Lawn, N.J.). Water for
HPLC was purified at point-of-use by a Milli-Q water system
(Millipore, Bedford, Mass.). Trifluoroacetic acid (TFA) was
purchased from Advanced ChemTech (Louisville, Ky.), (+)-Biotin,
di-t-butyl dicarbonate, diethylenetriamine, ethylenediamine, and
iodoacetic anhydride are from Aldrich Chemical Co. (Milwaukee,
Wis.), and 4-Fluoro-7-nitrobenzofurazan (NBD-F) is from Dojindo
Molecular Technologies, Inc. (Gaithersburg, Md.). All other
reagents were used as received. Unless otherwise noted, all
non-aqueous reactions were carried out under Argon atmosphere with
oven-dried glassware.
[0116] For spectral analyses, NMR spectra were recorded on Bruker
spectrometers: AC-200 (200 MHz), AC-300 (300 MHz) or AC-500 (500
MHz). In rare cases, NMR data had to be omitted for proteins that
are likely to be buried under the NMR peak from the solvent. Mass
spectral data was obtained using Bruker Daltonics (Bremen, Germany)
Esquire electrospray-ion trap mass spectrometer.
[0117] For chromatography, silica gel flash chromatography was
performed using 230-400 mesh silica gel 60 (Merck, Darmstadt,
Germany). Thin-Layer chromatography was performed on silica 60 with
F254, aluminum-backed plates (Merck). Reversed-phase HPLC
separations were performed using a Vydac (Hisperia, Calif.)
semi-preparative column (up to 5 mg total loading, 10 .mu.m
packing, 10 mm.times.250 mm, catalog no. 218TP1010) at 4 mL/min, or
a preparative column (up to 300 mg total loading, 10 .mu.m packing,
20 mm.times.250 mm, catalog no 218TP1022) at 6 mL/min.
[0118] Compound 1. (+)-Biotin (3 g, 12.3 mmol) was dissolved in
warm anhydrous DMF (60 mL). After the reaction was cooled to room
temperature, TEA (3.2 mL, 24.6 mmol) was added, followed by
tetrafluorophenyl trifluoroacetate (4.85 g, 18.5 mmol, prepared
according to the literature). After being stirred for 30 minutes at
room temperature, the solvent was removed under reduced pressure.
The residue was crystallized with diethyl ether, and the product
filtered (compound 1). The solid was dried under vacuum to afford
3.9 g (81%) of a white solid. .sup.1H-NMR (300 MHz, DMSO-d.sup.6)
.delta.7.9 (m, 1H); 6.4 (2H, d); 4.2 (2H, dt); 3.2 (m, 1H);
2.75-2.9 (m, 3H), 2.6 (1H, d); 1.4-1.8 (m, 6H).
[0119] Compound 2. To a round bottom flask containing
diethylenetriamine (5.5 g, 51 mmol) at 0.degree. C. was transferred
via cannula a solution of compound 1 (2 g, 5.10 mmol) in DMF (40
mL), also at 0.degree. C. After being stirred for 30 min at
0.degree. C., solvent was removed under reduced pressure. The
resulting residue was crystallized with ethyl ether (100 mL),
filtered, and washed with acetone (50 mL). The solid was left under
vacuum overnight, and 1.2 g (71%) of the desired product was
obtained (compound 2). .sup.1H-NMR (300 MHz, DMSO-d.sup.6)
.delta.6.4 (2H, d); 4.2 (2H, dt); 3.2 (m, 3H); 3.0-2.4 (m, 8H); 2.1
(t, 2H), 1.2-1.7 (m, 6H). ESI-MS (M+H.sup.+).sup.+: 330.4,
(M+2H.sup.+).sup.2+: 659.4.
[0120] Compound 3. To a round bottom flask containing a solution of
compound 2 (300 mg, 0.91 mmol) in DMF (30 mL) at 0.degree. C. was
slowly added a solution of di-t-butyl dicarbonate (200 mg, 0.91
mmol) in DMF (2 mL). After being stirred for an hour at 0.degree.
C., the ice bath was removed, and the reaction was left stirring
overnight at room temperature. The solvents were concentrated under
reduced pressure without heating. A mixture of ethyl ether and
methylene chloride was added to the residue, and the resulting
solution was stirred for 3-4 h. The final product was filtered, and
dried under vacuum to afford a white solid (compound 3) (290 mg,
74.4%). .sup.1H-NMR (300 MHz, DMSO-d.sup.6) .delta.6.4 (2H, d); 4.2
(2H, dt); 3.2 (3H, m); 3.0-2.4 (m, 8H); 2.1 (m, 2H), 1.2-1.7 (m,
15H). ESI-MS (M+H.sup.+).sup.+: 430.5.
[0121] Compound 4. To a vial containing a solution of compound 3
(130 mg, 0.30 mmol) in DMF (3 mL) was added a solution of
5-bromomethyl-1-(1-hydro- xyethyl)-2-nitrobenzene (prepared
according to the literature method, the material used also
contained 1 part in 4.6 of 5-methyl-1-(1-hydroxyethyl)-
-2-nitrobenzene)(160 mg) in DMF (1 mL), followed by TEA (86 .mu.L,
0.62 mmol). The reaction mixture was stirred at room temperature
for 7 days, and then placed in a shaker at 37.degree. C. for 2
days. The solvents were removed in a speed-vacuum apparatus, and
the reaction mixture was injected onto the HPLC column. Preparative
C.sub.18 column, detector .lambda.=240 nm, A=H.sub.2O with 0.08%
TFA, B=acetonitrile (MeCN) with 0.0806 TFA, gradient: 0'-20': 20%
B; 20'-80': 100% B. Product (compound 4)(50 mg, 27%) came out at
44% B. .sup.1H-NMR (300 MHz, MeOH-d.sup.4) .delta.8.05 (1H, s); 8.0
(1H, d); 7.7 (1H, d); 5.35 (1H, q); 4.5 (1H, dd); 4.25 (1H, dd);
3.6 (2H, bs); 3.45-3.25 (m, 8H); 3.2 (m, 1H), 2.95(1H, dd); 2.7(1H,
d); 2.2 (2H, t); 1.8-1.25 (m, 6H); 1.5 (3H, d); 1.4 (9H, s). ESI-MS
(M+H.sup.+).sup.+: 609.5, (M+Na.sup.+).sup.+: 631.5.
[0122] Compound 5. To a vial containing a solution of compound 4
(70 mg, 0.12 mmol) in a 1:1.75 mixture of DMF: CH.sub.2Cl.sub.2
(total volume of 550 .mu.L) was added a solution of carbonyl
diimidazole (28 mg, 0.17 mmol) in CH.sub.2Cl.sub.2 (850 .mu.L).
After being stirred at room temperature for 2 h in the presence of
4 angstrom molecular sieves, ethylenediamine (32 .mu.L, 0.23 mmol)
was added. After an additional 1.5 h, the solvent was removed in a
speedvacuum apparatus. The residue was injected onto the HPLC
column. Preparative C.sub.18 column, .lambda.=240 nm, A=H.sub.2O
with 0.08% TFA, B=MeCN with 0.08% TFA, gradient: 0'-20': 20% B;
20'-80': 100% B. Product (compound 5) as TFA salt (43.5 mg, 47%)
came out at 40% B. First intermediate: ESI-MS (M+H.sup.+).sup.+:
703.6, (M+Na.sup.+).sup.+: 725.6; Final product 5: ESI-MS
(M+H.sup.+).sup.+: 695.7, (M+Na.sup.+).sup.+: 717.7.
[0123] Compound 6. To a vial containing compound 5 (43.5 mg, 0.054
mmol) in THF (1 mL) in the presence of 4 angstrom molecular sieves,
was added TEA (6 .mu.L, 0.054 mmol), followed by a solution of
iodoacetic anhydride (21 mg, 0.059 mmol) in THF (1 mL). After
stirring for 1.5 h at room temperature, an excess of 1 equivalent
of TEA and 0.5 equivalents of iodoacetic anhydride were added to
the reaction mixture. After an additional 1.5 h at room
temperature, the solvents were removed in a speed-vacuum apparatus.
The residue was injected onto the HPLC column. Preparative C.sub.18
column, .lambda.=240 nm, A=H.sub.2O with 0.08% TFA, B=MeCN with
0.08% TFA, gradient: 0'-20': 20% B; 20'-80': 100% B. Product (23.4
mg, 55%) came out at 49% B (compound 6). ESI-MS (M+H.sup.+).sup.+:
863.7, (M+Na.sup.+).sup.+: 885.6
[0124] Compound 7. To a vial containing a solution of compound 6
(23.4 mg, 0.027 mmol) in CHCl.sub.3 (0.4 mL) was added TFA (80
.mu.L). The reaction mixture was stirred for 1.5 h at room
temperature. Solvent was removed using a speed-vacuum apparatus.
Methanol (MeOH) was added, and the resulting solution was
concentrated in the speed-vacuum apparatus. This procedure was
repeated 3 more times to remove excess TFA. The TFA salt of the
product (compound 7) was obtained in 98% yield (23.4 mg) as oil.
ESI-MS (M+H.sup.+).sup.+: 763.5.
[0125] Compound 8. To a vial containing compound 7 (10 mg, 0.011
mmol) was added a solution of NBD-F (7.2 mg, 0.027 mmol) in DMF
(640 .mu.L), followed by Hunnig's base (8 .mu.L, 0.046 mmol). After
being stirred at room temperature for 1 hr in the presence of 4
angstrom molecular sieves, the solvents were removed in a
speed-vacuum apparatus. The residue was injected onto the HPLC
column. Preparative C.sup.18 column, .lambda.=500 nm, A=H.sub.2O,
B=MeCN, gradient: 0'-20': 20% B; 20'-80': 100% B. An orange solid
(compound 8)(4.6 mg, 43.8%) came out at 51% B. .sup.1H-NMR (500
MHz, MeOH-d.sup.4) .delta.8.43 (1H, d); 7.6 (2H, bs); 7.45 (1H, d);
6.16 (1H, bs); 6.05 (1H, q); 36.8 (2H, bs); 3.65 (2H, m); 3.25-3.15
(m, 6H); 2.96-2.88 (6H, m); 2.8 (1H, t);. 2.71(1H, d); 2.22 (2H,
t); 1.8-1.55 (4H, m); 1.5 (3H, d); 1.42 (12H, m). ESI-MS
(M+H.sup.+).sup.+: 926.6, (M+Na.sup.+).sup.+: 948.7.
[0126] Other exemplary VICAT reagents can be readily synthesized by
the route shown in FIG. 2. Briefly, biotin p-nitrophenyl ester is
reacted with excess diethylenetriamine (both from Aldrich;
Milwaukee Wis.) to give the mono-amide, which is purified by rapid
silica gel chromatography using a simple 2-step solvent elution
procedure. Reaction of an excess of this mono-amide with
5-carboxytetramethylrhodamine succinimidyl ester (Molecular Probes
Inc.; Eugene Oreg.) gives the diamide, which is purified on a
reverse-phase HPLC column. Both the triamine linker and the
biotinylating reagent are inexpensive. Thus, the synthesis can be
performed using the short synthetic route shown in FIG. 2, with the
use of excess reagents (triamine in step 1 and mono-amide in step
2). Alternatively, a longer synthetic route involving linker
protection and deprotection steps can be used.
[0127] The diamide is reacted with
1-(4-bromomethyl-2-nitrophenyl)-ethan-2- -ol, which is prepared in
3 steps from commercially available 5-methyl-2-nitro-benzoic acid
(Aldrich) (Senter et al, Photochem. Photobiol. 42:231-237 (1988)).
This alkylation occurs preferentially on the secondary amine rather
than on the carboxylate of the tetramethylrhodamine moiety. Based
on analogous chemistry (Olejnik et al., supra, 1995), the benzyl
alcohol moiety is reacted with N,N'-disuccinimidyl carbonate
(Aldrich), and the active carbonate ester is reacted with
ICH.sub.2CONHCH.sub.2CH.sub.2NH.sub.2 (prepared by reaction of
BOC-ethylenediamine with iodoacetyl chloride followed by treatment
with trifluoroacetic acid) to give the desired VICAT reagent.
[0128] Additional exemplary VICAT reagents can be readily
synthesized by the route shown in FIG. 4B. The VICAT reagent shown
in FIG. 4 is an exemplary VICAT reagent having a radioactive
detectable moiety. The .sup.14C-acetyl-VICAT reagent is synthesized
(see FIG. 4B) in a manner substantially similar to that described
above for other VICAT reagents. Briefly, compound 1 is reacted with
ethylenediamine in DMF, followed by reaction with
5-bromomethyl-1-(1-hydroxyethyl)-2-nitrobenzene in DMF to generate
compound 2. Compound 2 is reacted with di-t-butyl dicarbonate in
DMF. To the product of this reaction is added carbonyldiimidazole
in DMF, followed by ethylenediamine. To the product of this
reaction is added iodoacetic anhydride, followed by TFA, to
generate compound 3. Compound 3 is dissolved in DMF, and sodium
1-[.sup.14C]acetate, TEA, and BOP
(Benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate) are added to generate compound 4
(.sup.14C-acetyl-VICAT).
[0129] The isotopically labeled VICAT reagents are prepared as
follows (FIGS. 1 and 2 insert). Di-.sup.13C-labeled oxalic acid
(Cambridge Isotopes; Andover Mass.) is distilled from
ethanol/H.sub.2SO.sub.4 to give diethyl oxalate, which is treated
with NH.sub.4OH in ethanol to give di-.sup.13C-labeled oxamate,
(.sup.13CONH.sub.2).sub.2 (Wilmes and Winnewisser, J. Labelled
Compd. Radiopharm. 31:1037-1040 (1992). The latter is reduced with
LiAlD.sub.4 (Aldrich) in tetrahydrofuran (THF) (Thomas, Deuterium
Labeling in Organic Chemistry, Appleton-Century-Crofts- , New York
(1971)) to give H.sub.2N.sup.13CD.sub.2.sup.13CD.sub.2NH.sub.2.
Repetition of this sequence of chemical reactions using
.sup.15NH.sub.4OH (Cambridge Isotopes)(excess .sup.15NH.sub.3 can
be fully recovered by bulb-to-bulb distillation) gives
H.sub.2.sup.15N.sup.13CD.sub.2.sup.13CD.- sub.2.sup.15NH.sub.2.
[0130] The labeled ethylenediamines is reacted with
di-t-butyldicarbonate to give the mono-BOC derivatives (Lochner et
al., Helv. Chim. Acta 81:2270-2281 (1998)). Di-.sup.13C-labeled
acetic acid (Cambridge Isotopes) is heated in
D.sub.2O/K.sub.2CO.sub.3 for 3-5 h at 120.degree. C. (Atkinson et
al., J. Am. Chem. Soc. 90: 498-499 (1968)) to give
.sup.13CD.sub.3.sup.13COOD, which is reacted with phosphorus and
Br.sub.2 to give Br.sup.13CD.sub.2.sup.13COBr (Ward, J. Chem. Soc.
1160-1165 (1922)). The labeled bromoacetyl bromide is reacted with
the BOC-protected ethylenediamine, followed by BOC removal with
trifluoroacetic acid, to give
Br.sup.13CD.sub.2.sup.13CO.sup.15NH.sup.13C-
D.sub.2.sup.13CD.sub.2.sup.15NH.sub.2, which is treated with KI in
acetone to give the desired
I.sup.13CD.sub.2.sup.13CO.sup.15NH.sup.13CD.sub.2.sup-
.13CD.sub.2.sup.15NH.sub.2 (12 atomic mass units (amu) higher than
the non-isotopically substituted compound). Likewise, the +6 amu
shifted compound
ICH.sub.2CONH.sup.13CD.sub.2.sup.13CD.sub.2NH.sub.2 is prepared in
the same way. All of these reactions proceed in near quantitative
yield and make use of relatively inexpensive heavy isotopelabeled
materials. The conversion of H.sub.2NCH.sub.2CH.sub.2NH.sub.2 to
ICH.sub.2CONHCH.sub.2CH.sub.2NH.sub.2 is obtained in 80% overall
yield with non-labeled materials.
[0131] This example describes a synthetic method for generating
VICAT reagents.
EXAMPLE II
[0132] Labeling, Detection and Cleavage of a Peptide Sample
[0133] This example describes a procedure for labeling protein
samples with VICAT reagents.
[0134] Cell or tissue protein are reduced, and cysteine-containing
proteins are tagged with a VICAT reagent. The sample is submitted
to proteolysis, for example, with trypsin, to generate the peptide
mixture. The proteolytic digestion can be carried out, for example,
overnight at a suitable temperature for the protease used, such as
22-37.degree. C. for trypsin. A defined amount of one or more
internal standards, peptides tagged with isotopically substituted
VICAT reagent, are added to the proteolytic digest. These internal
standards are readily prepared by solid-phase synthesis of the
peptides of interest (matching in sequence to the peptides
generated by proteolysis of the proteins of interest) and reacting
the cysteine of these synthetic peptides with the isotopically
heavy VICAT reagent.
[0135] The peptide mixture derived from the biological sample is
subjected to isoelectric focusing (TEF) in a polyacrylamide gel
strip. An adjacent IEF strip is loaded with the same internal
standard that was added to the complex protein mixture. Both IEF
strips are developed side-by-side, and visual inspection of the
strip containing internal standard alone reveals the precise
locations of the chemically identical VICAT-tagged peptide in the
adjacent IEF strip containing the complex peptide mixture. The
appropriate region of this latter strip is sliced from the strip,
and peptides in these slices are eluted with suitable solvents, for
example, 10 mM ammonia, 30% CH.sub.3CN. Other elution solvents can
be used, as desired, for example, aqueous buffer with detergent,
chaotropic agents, and the like, or organic solvents, which can be
used to elute peptides having low solubility in aqueous solution.
Elution yields can be monitored by the fluorescence of
VICAT-peptide conjugates. The eluted sample mixture is neutralized,
if desired, and bound to streptavidin agarose. The VICAT-labeled
and bound peptide is released by photocleavage from the solid phase
matrix, resulting in free labeled peptide, also called
NAMETAG-peptide.
[0136] Alternatively, a relatively large amount of one or more
internal standards can be chromatographed in a single IEF strip
with the sample of interest. Large amounts of these internal
standards are provided so that they can be visualized above a
background of VICAT-tagged, sample-derived peptides. These
visualized markers are tagged with, for example, the +12 VICAT
reagent so they can be distinguished from the internal standard
bearing, for example, a +6 VICAT tag and the peptide coming from
the example bearing, for example, the +0 VICAT tag.
[0137] The eluted tagged-peptides are subjected to photocleavage to
cleave the VICAT reagent from the peptide, generally so that most
of the VICAT reagent is cleaved. Photocleavage results in only a
small fragment, which bears the isotopic substitution, being
retained on the cysteine SH of the peptide. The resulting peptides
are analyzed, for example, using tandem ESI-MS. Because the complex
protein mixture also contains a known amount of peptide standard
tagged with isotopically substituted VICAT reagent, integration of
the ion signals from the heavy and light peptide conjugates allows
accurate quantification of the peptide of interest (see Gygi et
al., supra, 1999). In addition, peptide identification can be
obtained by mass spectrometric sequencing of the ESI-produced ions
by collision-induced dissociation (CID). Other mass spectrometric
methods can also be used for the detection, quantification and
identification of the tagged peptides, based on the fact that the
precise mass and sequence of the tagged peptide are known.
[0138] Using the above described method with VICAT reagents that
contain a fluorescent or chromophoric detectable group, all
cysteine-containing peptides in the complex cell-derived mixture
are made visible by tagging with the VICAT reagent. However, visual
localization is generally carried out only for the IEF strip that
bears the standard alone. Using chemically identical,
fluorophore-containing tags for chromatographic marker, internal
standard, and sample-derived peptides ensures identical
chromatographic behavior. In addition to using adjacent IEF strips
run side-by-side, a single strip containing two sample lanes can
also be used. Furthermore, the VICAT-labeled standard can be added
directly into the sample as an internal standard, particularly when
differential isotope VICAT reagents are available, such as the +0,
+6, and +12 reagents, as described above. The differentially
labeled peptides are expected to co-migrate since it has previously
been shown that ICAT-labeled proteins precisely co-migrate in
two-dimensional gels (IEF/SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis))(Smolka et al., Mol. Cell.
Proteomics 1:19-29 (2002)).
[0139] Using the above described method with VICAT reagents that
contain a radioactive detectable group, all cysteine-containing
peptides in the complex cell-derived mixture are made visible by
tagging with the VICAT reagent in a substantially similar manner to
that described above for fluorescent or chromophoric detectable
groups. For example, standard and sample can be run in parallel
gels or parallel lanes on the same gel. The use of a radioactive
detectable group allows the convenient addition of the standard to
the same gel strip or even directly into the sample being analyzed,
with localization of a particular sample molecule determined by
detection of the radiolabel of the corresponding standard.
[0140] The above-described method using an internal standard can be
used to determine the absolute amount of sample peptide in the
biological sample. To calculate the amount of sample protein from
the amount of peptide, the efficiency of proteolytic generation of
the peptide is determined. Alternatively, the amount of
sample-derived peptide can be compared to that of a control sample,
if desired.
[0141] This example describes a general protocol for the use of a
VICAT reagent to label proteins in a sample.
EXAMPLE III
[0142] Isoelectric Focusing and ESI-MS Analysis of Peptides
[0143] This example describes the analysis of peptides labeled with
a VICAT reagent using isolectric focusing and ESI-MS.
[0144] The peptide mixture consisting of the tagged peptides from
the protein sample mixture and the differentially isotopically
tagged internal standard are often very complex and need to be
separated prior to ESI-MS analysis. Any of a number of peptide
separation methods can be applied to the sample, for example,
chromatography, electrophoresis, and the like. Among the multitude
of mature peptide separation methods available, isoelectric
focusing (IEF) in polyacrylamide gels with immobilized pH gradients
is particularly useful for the following reasons: i) The method is
robust, relatively inexpensive, highly reproducible and easily
multiplexed (Righetti et al., Methods. Enzymol. 270:235-55 (1996)).
IEF strips covering different pH ranges are commercially available
(for example, Pharmacia-Amersham Biotech; Piscataway N.J.); ii) The
method has immense separation power (Righetti et al., supra, 1996),
and the pH gradient, and therefore the range of maximal separation,
can be tuned, as desired for particular experimental applications;
iii) The pI of peptides can be reasonably well calculated from
their amino acid sequences (Bjellqvist et al., Electrophoresis
14:1023-1031 (1993)); thus, the migration position of a specific
target peptide-VICAT conjugate in the gel can be predicted,
allowing rational choice of the pH range of the IEF used values.
The tetramethylrhodamine group of the VICAT. reagent will be
zwitterionic when the pH equals the pI for most peptides, and thus
the peptide and VICAT-peptide conjugate will have similar pI
values. Furthermore, the pI value of the VICAT-peptide conjugate
can be calculated using the pKa value of the COOH group of the
tetramethylrhodamine; and vi) Peptide recovery is achieved in high
yield simply by soaking the gel segment in an appropriate solvent
such as an aqueous buffer.
[0145] For analysis of peptide samples by IEF, a peptide sample
generated by tryptic digestion, or other suitable protease
digestion, of the VICAT-labeled protein sample essentially as
described in Example II (see also Gygi et al., supra, 1999).
Internal standard peptide(s) are added to the VICAT-labeled digest
with soluble carrier ampholytes to a final concentration of 0.5%
and applied to a rehydration cassette (Pharmacia-Amersham Biotech).
The gel is allowed to swell for 4 h. The gel strips are transferred
to the electrophoresis chamber and focused at a final voltage of
8000V for a total of 115,000 volt-hours. The voltage is slowly
ramped up to avoid peptide precipitation and local overheating of
the gel. A second identical gel strip is loaded with the external
tagged peptide standard(s) alone for the localization of the target
peptide(s) in the gel. The gel strips are subjected either to
visual inspection or to fluorescence imaging, if needed for
increased sensitivity. The gel segments containing the target
peptide(s) are extracted and the isolated peptides are bound to the
affinity matrix. The affinity isolated peptides are subjected to
photocleavage to remove the fluorescent tag and release the
NAMETAG-peptides having the isotopic label. The peptide-NAMETAG
conjugates are analyzed by combined microbore-HPLC/ESI-MS using the
standard protocols and software tools developed for quantitative
protein profiling by the ICAT method (Han et al., Nat. Biotechnol.
19:946-951 (2001); Gygi et al., supra, 1999).
[0146] If the affinity reagent is avidin, the extracted peptides
are bound to streptavidin modified agarose. beads or streptavidin
modified magnetic beads using standard protocols and washed.
NAMETAG peptides are recovered from the solid support by photo
cleavage of the linker. The beads re re-suspended in of 0.2 M Tris,
pH 8.0, 10 mM EDTA with 2% .beta.-mercaptoethanol to prevent
methionine oxidation during photo-cleavage. Light from a Blak-Ray
longwave UV lamp (VWR) is filtered by 10% of copper (II) sulfate
solution (1 cm path length) and used to illuminate the beads for 2
hours. Beads are occasionally agitated to ensure uniform light
illumination. The recovered peptides are analyzed by LC-MS/MS using
the standard protocols and software tools developed for
quantitative protein profiling (Gygi et al., supra, 1999; Han et
al., Nat. Biotechnol. 19:946-951 (2001)).
[0147] This example describes protocols for IEF and ESI-MS analysis
of VICAT-labeled peptides.
EXAMPLE IV
[0148] Dystrophin Analysis Using VICAT Reagents
[0149] This example describes the use of the VICAT technology for
the quantification of dystrophin and its structural variants, which
is clinically relevant for the diagnoses of Duchenne's and Becker's
muscular dystrophy.
[0150] The distribution of pI values for the full set of
cysteine-containing, tryptic peptides derived from the human
proteome is calculated. With current genome annotation,
.about.33,000 proteins are predicted, giving .about.230,000
cysteine-containing, tryptic peptides. It is expected that far
fewer proteins will be expressed in any given cell type. The
distribution of pI values is not uniform; clusters of up to a few
thousand peptides exist. Thus, it is useful to search for those
dystrophin-derived peptides that will migrate in the IEF strip in a
noncrowded region. There are 8 dystrophin-derived,
cysteine-containing, tryptic peptides that will migrate in
relatively non-crowded regions containing <50 peptides. Also,
the pH gradient in the crowded regions can be flattened, providing
better separation.
[0151] The VICAT method allows selective detection of segments of
dystrophin, which is useful for the diagnosis of variants of
muscular dystrophy caused by dystrophin deletions. The amount of
dystrophin present in total skeletal muscle protein can also be
determined. Initial studies are carried out with the crude surface
membrane fraction prepared from skeletal muscle using a simple
3-step differential centrifugation procedure (Ohlendieck et al., J.
Cell Biol. 112:135-148 (1991)). Approximately 1% of the total
protein in this fraction is dystrophin, whereas this protein
constitutes 0.002% of total skeletal muscle protein (Ohlendieck and
Campbell, FEBS Lett. 283:230-234 (1991)). A 100 mg sample of
skeletal muscle obtained by needle biopsy contains .about.0.5 .mu.g
of dystrophin (.about.1 pmol), which is more than sufficient for
analysis. For example, 10-50 fmol of peptide can be routinely
detected when this amount is applied to microbore-HPLC/ESI-MS
system.
[0152] For analysis of dystrophin in muscle sample, proteins in the
membrane fraction are solubilized by boiling in 50 mM Tris-HCl, pH
8.3, 5 mM EDTA, and either 0.5% SDS or 0.05% SDS/6 M urea. These
buffers have been shown to work well for ICAT analysis of
microsomal proteins (Han et al., Nat. Biotechnol. 19:946-951
(2001); Smolka et al., Anal. Biochem. 297:25-31 (2001)). Protein
disulfides are reduced by treatment with 5 mM tributyl phosphine
for 30 min at 37.degree. C. (Smolka et al., supra, 2001), and
cysteines are tagged with 200 nM to 1 mM VICAT reagent, depending
on the amount of peptides in the sample to be analyzed, for 90 min
at room temperature. The sample is diluted 10-fold to reduce the
concentration of denaturants, a known amount of internal standard
(for example, synthetic dystrophin peptide tagged with VICAT(+6))
is added, and the sample is treated with trypsin overnight at
37.degree. C. VICAT-peptide conjugates can be isolated using
solid-phase, monomeric avidin as described previously for ICAT
reagents (Gygi et al., supra, 1999). IEF and microbore-HPLC/ESI-MS
are carried out as described above. If peptide sample and IEF
marker are run in separate lanes of the same IEF strip, the
VICAT(+12) reagent can optionally be used to prepare the IEF marker
in case some of it diffuses into the adjacent IEF lane containing
the peptide mixture. Alternatively, the VICAT-labeled peptide
standards are added to the sample molecules for resolution in a
single IEF lane.
[0153] Alternatively, following reaction of sample peptides with a
VICAT reagent, salt and detergent are removed by ion exchange
chromatography, such as cation exchange chromatography, and step
elution. This step can also serve to exchange the buffer
composition for further manipulations. VICAT-labeled peptides in
the sample mixture are resolved by IEF. The location of
VICAT-labeled sample peptides are visualized using VICAT labeled
standard peptides resolved in parallel or as an internal standard
added to the sample mixture. Resolved VICAT-labeled peptides of
interest are absorbed onto streptavidin beads. Photocleavage is
carried out on the affinity isolated peptides, resulting in release
of NAMETAG-peptide conjugate(s), which contain the isotope tag. The
released NAMETAG-peptide conjugate(s) are analyzed by LC-MS/MS.
Digestion with a protease such as trypsin can optionally be
included at various steps, such as before or after reacting with
the VICAT reagent, before or after resolving sample molecules, or
before or after binding to the affinity resin.
[0154] The quantification of dystrophin using VICAT reagents can be
carried out using mouse skeletal muscle. Frozen tissue is
commercially available (Pel-Freeze Inc.; Rogers Ariz.). Of the 20
cysteine-containing, tryptic peptides of >5 amino acids derived
from dystrophin, 16 have identical sequences for mouse and human
proteins, and 4 have nearly identical sequences. Skeletal muscle
from the mdx mouse, which lacks dystrophin, can also be used
(Sicinski et al., Science 244:1578-1580 (1989)). After validation
of the VICAT assay using mouse tissue, human muscle samples are
obtained using Institutional Review Board approved protocols and
analyzed as described above.
[0155] This example describes the analysis of dystrophin using a
VICAT-reagent.
[0156] Throughout this application various publications have been
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference in this application
in order to more fully describe the state of the art to which this
invention pertains. Although the invention has been described with
reference to the examples provided above, it should be understood
that various modifications can be made without departing from the
spirit of the invention.
* * * * *